Protein expression from multiple nucleic acids

ABSTRACT

The current invention reports a method for the recombinant production of a secreted heterologous immunoglobulin in a CHO cell comprising the following steps: i) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, and virus free, ii) providing a vector comprising a prokaryotic origin of replication, a first nucleic acid conferring resistance to a prokaryotic selection agent, a second nucleic acid encoding the heavy chain of said heterologous immunoglobulin, a third nucleic acid encoding the light chain of said heterologous immunoglobulin, a fourth nucleic acid conferring resistance to a eukaryotic selection agent, iii) transfecting said CHO cell, wherein said transfecting comprises a) transfecting said CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a first eukaryotic selection agent, b) selecting a CHO cell by growth in cultivation medium containing said first eukaryotic selection agent, c) transfecting said selected CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a second eukaryotic selection agent different to said first eukaryotic selection agent, d) selecting a CHO cell by selected growth in cultivation medium containing said first and said second eukaryotic selection agent, iv) cultivating said transfected CHO cell in a medium in the presence of said first and second eukaryotic selection agent, under conditions suitable for the expression of said second, and third nucleic acid, and v) recovering said secreted heterologous immunoglobulin from the cultivation medium.

This application is the National Stage of International Application No.PCT/EP2008/008523, filed Oct. 9, 2008, which claims the benefit of EP07019999.7 filed Oct. 12, 2007, which is hereby incorporated byreference in its entirety.

The current invention is in the field of polypeptide production. Moreprecisely it is reported the production of an immunoglobulin in amammalian cell whereby the mammalian cell is transfected with differentvectors each comprising an expression cassette for the immunoglobulin ofinterest.

BACKGROUND OF THE INVENTION

Expression systems for the production of recombinant polypeptides arewell-known in the state of the art and are described by, e.g., Marino,M. H., Biopharm. 2 (1989) 18-33; Goeddel, D. V., et al., MethodsEnzymol. 185 (1990) 3-7; Wurm, F., and Bernard, A., Curr. Opin.Biotechnol. 10 (1999) 156-159. Polypeptides for use in pharmaceuticalapplications are preferably produced in mammalian cells such as CHOcells, NS0 cells, SP2/0 cells, COS cells, HEK cells, BHK cells, PER.C6®cells, or the like. The essential elements of an expression plasmid area prokaryotic plasmid propagation unit, for example for E. coli,comprising a prokaryotic origin of replication and a prokaryoticselection marker, an eukaryotic selection marker, and one or moreexpression cassettes for the expression of the structural gene(s) ofinterest each comprising a promoter, a structural gene, and atranscription terminator including a polyadenylation signal. Fortransient expression in mammalian cells a mammalian origin ofreplication, such as the SV40 Ori or OriP, can be included. As promotera constitutive or inducible promoter can be selected. For optimizedtranscription a Kozak sequence may be included in the 5′ untranslatedregion. For mRNA processing, in particular mRNA splicing andtranscription termination, mRNA splicing signals, depending on theorganization of the structural gene (exon/intron organization), may beincluded as well as a polyadenylation signal.

Expression of a gene is performed either as transient or as permanentexpression. The polypeptide(s) of interest are in general secretedpolypeptides and therefore contain an N-terminal extension (also knownas the signal sequence) which is necessary for the transport/secretionof the polypeptide through the cell into the extracellular medium. Ingeneral, the signal sequence can be derived from any gene encoding asecreted polypeptide. If a heterologous signal sequence is used, itpreferably is one that is recognized and processed (i.e. cleaved by asignal peptidase) by the host cell. For secretion in yeast for examplethe native signal sequence of a heterologous gene to be expressed may besubstituted by a homologous yeast signal sequence derived from asecreted gene, such as the yeast invertase signal sequence, alpha-factorleader (including Saccharomyces, Kluyveromyces, Pichia, and Hansenulaα-factor leaders, the second described in U.S. Pat. No. 5,010,182), acidphosphatase signal sequence, or the C. albicans glucoamylase signalsequence (EP 0 362 179). In mammalian cell expression the native signalsequence of the protein of interest is satisfactory, although othermammalian signal sequences may be suitable, such as signal sequencesfrom secreted polypeptides of the same or related species, e.g. forimmunoglobulins from human or murine origin, as well as viral secretorysignal sequences, for example, the herpes simplex glycoprotein D signalsequence. The DNA fragment encoding for such a presegment is ligated inframe to the DNA fragment encoding a polypeptide of interest.

Today CHO cells are widely used for the expression of pharmaceuticalpolypeptides, either at small scale in the laboratory or at large scalein production processes. Due to their wide distribution and use thecharacteristic properties and the genetic background of CHO cells iswell known. Therefore, CHO cells are approved by regulatory authoritiesfor the production of therapeutic proteins for application to humanbeings.

In EP 0 569 678 are reported double transfectants of MHC genes ascellular vaccines for immunoprevention of tumor metastasis. WO 97/08342reports an improved method for measuring the activity of a promotersequence in a mammalian cell using a reporter gene. The use of anti-RhoAand anti-RhoC siRNAs in order to inhibit specifically RhoA or RhoCsynthesis is reported in WO 2005/113770. A method for the recombinantproduction or expression of eukaryotic alkaline phosphatase mutant inyeast cells is reported in U.S. Pat. No. 7,202,072. WO 2001/038557reports a method of screening multiply transformed cells usingbicistronic expression of fluorescent proteins. A method for producingrecombinant eukaryotic cell lines expressing multiple proteins or RNAsof interest is reported in WO 1999/47647. Systems, including methods,compositions, and kits, for transfection of cells with transfectionmaterials using coded carriers are reported in WO 2003/076588. In U.S.Pat. No. 5,089,397 is reported an expression system for recombinantproduction of a desired protein comprising CHO cells transformed with aDNA sequence having the desired protein coding sequence under thecontrol of the human metallothionein-II promoter. A method for producingrecombinant proteins is reported in US 2003/0040047. Lamango et al.(Lamango, N. S., et al., Arch. Biochem. Biophys. 330 (1996) 238-250)report the dependency of the production of prohormone convertase 2 fromthe presence of the neuroendocrine polypeptide 7B2. The transfection ofa BPV-1-based expression vector into cells harboring unintegratedreplicating BPV-1 genomes is reported by Waldenstroem, M., et al., Gene120 (1992) 175-181. U.S. Pat. No. 4,912,038 reports methods and vectorsfor obtaining canine and human 32K alveolar surfactant protein. In WO89/10959 are reported recombinant DNA techniques and the expression ofmammalian polypeptides in genetically engineered eukaryotic cells. Arepeated co-transfer of an expression vector for human growth hormoneand an expression vector for a selection marker gene is reported in DD287531.

SUMMARY OF THE INVENTION

A first aspect of the current invention is a method for the recombinantproduction of a heterologous immunoglobulin which is secreted to thecultivation medium in a CHO cell comprising:

-   a) providing a CHO cell, which is    -   adapted to growth in suspension culture,    -   adapted to growth in serum-free medium,    -   mycoplasm free, and    -   optional virus free,-   b) providing a nucleic acid comprising    -   a prokaryotic origin of replication,    -   a first nucleic acid sequence conferring resistance to a        prokaryotic selection agent,    -   a second nucleic acid sequence encoding the heavy chain of said        heterologous immunoglobulin, and/or a third nucleic acid        sequence encoding the light chain of said heterologous        immunoglobulin,    -   whereby a first transfection vector is provided which comprises        said provided nucleic acid, which comprises said first as well        as said second and/or third nucleic acid, and an additional        fourth nucleic acid sequence conferring resistance to a first        eukaryotic selection agent, and    -   whereby a second transfection vector is provided which comprises        said provided nucleic acid, which comprises the identical first        as well as second and/or third nucleic acid as that/those in        said provided nucleic acid contained in the first transfection        vector, and an additional fourth nucleic acid sequence        conferring resistance to a second eukaryotic selection agent,        which is different from the fourth nucleic acid in said first        transfection vector, whereby said second eukaryotic selection        agent is different from said first eukaryotic selection agent,-   c) transfecting said provided CHO cell and selecting said    transfected CHO cell with said transfection vectors of step b),    wherein said transfecting and selecting comprises the following    steps in the following order:    -   (i) transfecting said CHO cell with said first transfection        vector,    -   (ii) selecting a CHO cell transfected in (i) by selected growth        in a cultivation medium containing said first eukaryotic        selection agent to which the first transfection vector confers        resistance,    -   (iii) transfecting said CHO cell selected in (ii) with said        second transfection vector,    -   (iv) selecting a CHO cell transfected in (iii) by selected        growth in a cultivation medium containing said first eukaryotic        selection agent, to which said first transfection vector confers        resistance, and containing said second eukaryotic selection        agent, to which said second transfection vector confers        resistance,-   d) cultivating said transfected and selected CHO cell of step c) in    a medium containing said first and second eukaryotic selection agent    under conditions suitable for the expression of said second and/or    third nucleic acid,-   e) recovering said secreted heterologous immunoglobulin from the    cultivation medium and thereby producing a heterologous    immunoglobulin in a CHO cell, which immunoglobulin is secreted to    the cultivation medium.

In one embodiment of the method according to the invention said CHO cellis a CHO K1 cell, or a CHO DG44 cell, or a CHO XL99 cell, or a CHO DXB11cell, or a CHO DP12 cell. In another embodiment the promoter employedfor the transcription of said second and third nucleic acids isdifferent from the promoter employed for the transcription of saidfourth nucleic acid. A further embodiment is that the promoter employedfor the transcription of said second and third nucleic acids is thesame. In one embodiment said promoter employed for the transcription ofsaid second and third nucleic acid is the CMV promoter. In anotherembodiment said promoter employed for the transcription of said fourthnucleic acid is the SV40 promoter. In one embodiment said heterologousimmunoglobulin is an anti-Aβ antibody. Exemplary anti-Aβ antibodies arereported e.g. in WO 2003/070760.

In one embodiment said selecting a transfected CHO cell in step c) (ii)and/or (iv) is by growth in cultivation medium without a selection agentfor 10 to 72 hours followed by selected growth in a cultivation mediumcontaining said first eukaryotic selection agent in case of (ii) or saidfirst and second eukaryotic selection agent in case of (iv).

In still a further embodiment the codon usage of said second and thirdnucleic acid is optimized for the translation in CHO cells. Also anembodiment is that said second and/or third nucleic acid contains anintronic nucleic acid sequence. Another embodiment comprises that saidfirst transfection vector and said second transfection vector differonly in the nucleic acid conferring resistance to said eukaryoticselection agent, i.e. in said fourth nucleic acid, and are otherwise atleast 95% identical based on the nucleic acid sequence. In anotherembodiment said transfection vectors differ each only in the nucleicacid conferring resistance to said first, second, and third eukaryoticselection agent.

In one embodiment said method further comprises:

after step b) a step b1):

-   b1) providing a nucleic acid comprising    -   a prokaryotic origin of replication,    -   a first nucleic acid sequence conferring resistance to a        prokaryotic selection agent,    -   a second nucleic acid sequence encoding the heavy chain of said        heterologous immunoglobulin, and/or a third nucleic acid        sequence encoding the light chain of said heterologous        immunoglobulin,    -   whereby a third transfection vector is provided which comprises        said provided nucleic acid, which comprises the identical frist        as well as second and/or third nucleic acid as that/those in        said provided nucleic acid contained in the first and second        transfection vector, and an additional fourth nucleic acid        sequence conferring resistance to a third eukaryotic selection        agent, which is different from the fourth nucleic acid in said        first and second transfection vector, whereby said third        eukaryotic selection agent is different from said first        eukaryotic selection agent and is also different from said        second eukaryotic selection agent,        and further comprises after step c) (iv) the following steps (v)        and (vi)    -   (v) transfecting said CHO cell selected in (iv) with said third        transfection vector,    -   (vi) selecting a CHO cell transfected in (v) by selected growth        in cultivation medium containing said first eukaryotic selection        agent to which the first transfection vector confers resistance        and said second eukaryotic selection agent to which the second        transfection vector confers resistance and said third eukaryotic        selection agent to which the third transfection vector confers        resistance,        and further said medium for cultivating said transfected CHO        cell in step d) comprises said first, second, and third        eukaryotic selection agent.

In one embodiment said selecting a CHO cell transfected in step c) (vi)is by growth in cultivation medium without a selection agent for 10 to72 hours followed by selected growth in a cultivation medium containingsaid first and second and third eukaryotic selection agent.

In another embodiment the method according to the invention comprises afurther step

-   f) purifying said recombinantly produced and recovered heterologous    immunoglobulin of step e) with one or more chromatographic steps.

One embodiment is that said step c) and said step d) are performed inthe same medium. Still another embodiment is that said medium is aserum-free medium, or a serum-free medium supplemented with definedanimal-derived components, or an animal-derived component free medium,or a protein-free medium, or a chemically defined medium, or a definedprotein-free medium. In a further embodiment in said step d) is saidcultivating in the presence of said eukaryotic selection agents in avolume of less than 500 liter and said cultivating is in the absence ofsaid eukaryotic selection agents in a volume of 500 liter or more,whereby said recovering said secreted heterologous immunoglobulin isfrom the cultivation medium without said eukaryotic selection agents. Ina further embodiment said cultivating in said step d) is comprisingsequential cultivations each with increasing cultivation volume up to apreset final cultivation volume, whereby the cultivations are performedin the presence of said eukaryotic selection agents up to a cultivationvolume of 1% (v/v) of the cultivation volume of the final cultivationand in the absence of said eukaryotic selection agents in a cultivationvolume of more than 1% (v/v) of the cultivation volume of the finalcultivation.

The productivity of said CHO cells is in one embodiment over 40generations not less than 70% and not more than 130% of the productivityafter 10 generations of cultivation as split-batch cultivation. Inanother embodiment is the productivity of said CHO cells over 60generations not less than 50% and not more than 150% of the productivityafter 10 generations of cultivation as split-batch cultivation. In stilla further embodiment is the productivity of said CHO cell at least 1.5g/l of said heterologous immunoglobulin within 21 days as fed-batchcultivation.

A second aspect of the current invention is a CHO cell obtainable withthe following method:

-   a) providing a CHO cell, which is    -   adapted to growth in suspension culture,    -   adapted to growth in serum-free medium,    -   mycoplasma free, and    -   optional virus free,-   b) providing a nucleic acid comprising    -   a prokaryotic origin of replication,    -   a first nucleic acid sequence conferring resistance to a        prokaryotic selection agent,    -   a second nucleic acid sequence encoding the heavy chain of a        heterologous immunoglobulin, and/or a third nucleic acid        sequence encoding the light chain of a heterologous        immunoglobulin,    -   whereby a first transfection vector is provided which comprises        said provided nucleic acid, which comprises said frist as well        as second and/or third nucleic acid, and an additional fourth        nucleic acid sequence conferring resistance to a first        eukaryotic selection agent, and    -   whereby a second transfection vector is provided which comprises        said provided nucleic acid, which comprises the identical frist        as well as second and/or third nucleic acid as that/those in        said provided nucleic acid contained in the first transfection        vector, and an additional fourth nucleic acid sequence        conferring resistance to a second eukaryotic selection agent,        which is different from the fourth nucleic acid in said first        transfection vector, whereby said second eukaryotic selection        agent is different from said first eukaryotic selection agent,-   c) transfecting said CHO cell, wherein said transfecting comprises    the following steps in the following order:    -   (i) transfecting said CHO cell with said first transfection        vector,    -   (ii) selecting a CHO cell transfected in (i) by selected growth        in cultivation medium containing a first eukaryotic selection        agent to which the first transfection vector confers resistance,    -   (iii) transfecting said CHO cell selected in (ii) with said        second transfection vector,    -   (iv) selecting a CHO cell transfected in (iii) by selected        growth in cultivation medium containing said first eukaryotic        selection agent to which the first transfection vector confers        resistance and said second eukaryotic selection agent to which        the second transfection vector confers resistance.

DETAILED DESCRIPTION OF THE INVENTION

Methods and techniques known to a person skilled in the art, which areuseful for carrying out the current invention, are described e.g. inAusubel, F. M., ed., Current Protocols in Molecular Biology, Volumes Ito III (1997), Wiley and Sons; Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989).

General chromatographic methods and their use are known to a personskilled in the art. See for example, Chromatography, 5^(th) edition,Part A: Fundamentals and Techniques, Heftmann, E. (ed), Elsevier SciencePublishing Company, New York, (1992); Advanced Chromatographic andElectromigration Methods in Biosciences, Deyl, Z. (ed.), ElsevierScience BV, Amsterdam, The Netherlands, (1998); Chromatography Today,Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, NewYork, (1991); Scopes, Protein Purification: Principles and Practice(1982); Sambrook, J., et al. (ed), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; or Current Protocols in Molecular Biology, Ausubel,F. M., et al. (eds), John Wiley & Sons, Inc., New York.

For the purification of recombinantly produced heterologousimmunoglobulins often a combination of different column chromatographysteps is employed. Generally a Protein A affinity chromatography isfollowed by one or two additional separation steps. The finalpurification step is a so called “polishing step” for the removal oftrace impurities and contaminants like aggregated immunoglobulins,residual HCP (host cell protein), DNA (host cell nucleic acid), viruses,or endotoxins. For this polishing step often an anion exchange materialin a flow-through mode is used.

Different methods are well established and widespread used for proteinrecovery and purification, such as affinity chromatography withmicrobial proteins (e.g. protein A or protein G affinitychromatography), ion exchange chromatography (e.g. cation exchange(carboxymethyl resins), anion exchange (amino ethyl resins) andmixed-mode exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi,M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

The term “amino acid” as used within this application denotes the groupof carboxy α-amino acids, which directly or in form of a precursor canbe encoded by a nucleic acid. The individual amino acids are encoded bynucleic acids consisting of three nucleotides, so called codons orbase-triplets. Each amino acid is encoded by at least one codon. Theencoding of the same amino acid by different codons is known as“degeneration of the genetic code”. The term “amino acid” as used withinthis application denotes the naturally occurring carboxy α-amino acidsand is comprising alanine (three letter code: ala, one letter code: A),arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G),histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys,K), methionine (met, M), phenylalanine (phe, F), proline (pro, P),serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr,Y), and valine (val, V).

A “nucleic acid” or a “nucleic acid sequence”, which terms are usedinterchangeably within this application, refers to a polymeric moleculeconsisting of individual nucleotides (also called bases) a, c, g, and t(or u in RNA), for example to DNA, RNA, or modifications thereof. Thispolynucleotide molecule can be a naturally occurring polynucleotidemolecule or a synthetic polynucleotide molecule or a combination of oneor more naturally occurring polynucleotide molecules with one or moresynthetic polynucleotide molecules. Also encompassed by this definitionare naturally occurring polynucleotide molecules in which one or morenucleotides are changed (e.g. by mutagenesis), deleted, or added. Anucleic acid can either be isolated, or integrated in another nucleicacid, e.g. in an expression cassette, a plasmid, or the chromosome of ahost cell. A nucleic acid is characterized by its nucleic acid sequenceconsisting of individual nucleotides.

To a person skilled in the art procedures and methods are well known toconvert an amino acid sequence, e.g. of a polypeptide, into acorresponding nucleic acid sequence encoding this amino acid sequence.Therefore, a nucleic acid is characterized by its nucleic acid sequenceconsisting of individual nucleotides and likewise by the amino acidsequence of a polypeptide encoded thereby.

A “polypeptide” is a polymer consisting of amino acids joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 20 amino acid residues may be referred to as “peptides”,whereas molecules consisting of two or more polypeptides or comprisingone polypeptide of more than 100 amino acid residues may be referred toas “proteins”. A polypeptide may also comprise non-amino acidcomponents, such as carbohydrate groups, metal ions, or carboxylic acidesters. The non-amino acid components may be added by the cell, in whichthe polypeptide is expressed, and may vary with the type of cell.Polypeptides are defined herein in terms of their amino acid backbonestructure or the nucleic acid encoding the same. Additions such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “immunoglobulin” encompasses the various forms ofimmunoglobulin structures including complete immunoglobulins andimmunoglobulin conjugates. The immunoglobulin employed in the currentinvention is preferably a human antibody, or a humanized antibody, or achimeric antibody, or a T cell antigen depleted antibody (see e.g. WO98/33523, WO 98/52976, and WO 00/34317). Genetic engineering ofantibodies is e.g. described in Morrison, S. L., et al., Proc. Natl.Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and5,204,244; Riechmann, L., et al., Nature 332 (1988) 323-327; Neuberger,M. S., et al., Nature 314 (1985) 268-270; Lonberg, N., Nat. Biotechnol.23 (2005) 1117-1125. Immunoglobulins may exist in a variety of formats,including, for example, Fv, Fab, and F(ab)₂ as well as single chains(scFv) or diabodies (e.g. Huston, J. S., et al., Proc. Natl. Acad. Sci.USA 85 (1988) 5879-5883; Bird, R. E., et al., Science 242 (1988)423-426; in general, Hood et al., Immunology, Benjamin N.Y., 2nd edition(1984); and Hunkapiller, T. and Hood, L., Nature 323 (1986) 15-16).

The term “complete immunoglobulin” denotes an immunoglobulin whichcomprises two so called light chains and two so called heavy chains.Each of the heavy and light chains of a complete immunoglobulin containsa variable domain (variable region) (generally the amino terminalportion of the polypeptide chain) comprising binding regions that areable to interact with an antigen. Each of the heavy and light chains ofa complete immunoglobulin comprises a constant region (generally thecarboxyl terminal portion). The constant region of the heavy chainmediates the binding of the antibody i) to cells bearing a Fc gammareceptor (FcγR), such as phagocytic cells, or ii) to cells bearing theneonatal Fc receptor (FcRn) also known as Brambell receptor. It alsomediates the binding to some factors including factors of the classicalcomplement system such as component (C1q). The variable domain of animmunoglobulin's light or heavy chain in turn comprises differentsegments, i.e. four framework regions (FR) and three hypervariableregions (CDR).

The term “immunoglobulin conjugate” denotes a polypeptide comprising atleast one domain of an immunoglobulin heavy or light chain conjugatedvia a peptide bond to a further polypeptide. The further polypeptide isa non-immunoglobulin peptide, such as a hormone, or growth receptor, orantifusogenic peptide, or complement factor, or the like. Exemplaryimmunoglobulin conjugates are reported in WO 2007/045463.

The term “heterologous immunoglobulin” denotes an immunoglobulin whichis not naturally produced by a mammalian cell or the host cell. Theimmunoglobulin produced according to the method of the invention isproduced by recombinant means. Such methods are widely known in thestate of the art and comprise protein expression in eukaryotic cellswith subsequent recovery and isolation of the heterologousimmunoglobulin, and usually purification to a pharmaceuticallyacceptable purity. For the production, i.e. expression, of animmunoglobulin a nucleic acid encoding the light chain and a nucleicacid encoding the heavy chain are inserted each into an expressioncassette by standard methods. Nucleic acids encoding immunoglobulinlight and heavy chains are readily isolated and sequenced usingconventional procedures. Hybridoma cells can serve as a source of suchnucleic acids. The expression cassettes may be inserted into anexpression plasmid(s), which is (are) then transfected into host cells,which do not otherwise produce immunoglobulins. Expression is performedin appropriate prokaryotic or eukaryotic host cells and theimmunoglobulin is recovered from the cells after lysis or from theculture supernatant.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e. at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

“Heterologous DNA” or “heterologous polypeptide” refers to a DNAmolecule or a polypeptide, or a population of DNA molecules or apopulation of polypeptides, that do not exist naturally within a givenhost cell. DNA molecules heterologous to a particular host cell maycontain DNA derived from the host cell species (i.e. endogenous DNA) solong as that host DNA is combined with non-host DNA (i.e. exogenousDNA). For example, a DNA molecule containing a non-host DNA segmentencoding a polypeptide operably linked to a host DNA segment comprisinga promoter is considered to be a heterologous DNA molecule. Conversely,a heterologous DNA molecule can comprise an endogenous structural geneoperably linked with an exogenous promoter.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

The term “cell” or “host cell” refers to a cell into which a nucleicacid, e.g. encoding a heterologous polypeptide, can be or istransfected. The term “cell” includes both prokaryotic cells, which areused for propagation of plasmids, and eukaryotic cells, which are usedfor the expression of a nucleic acid and production of the encodedpolypeptide. In one embodiment, the eukaryotic cells are mammaliancells. In another embodiment the mammalian cell is a CHO cell,preferably a CHO K1 cell (ATCC CCL-61 or DSM ACC 110), or a CHO DG44cell (also known as CHO-DHFR[−], DSM ACC 126), or a CHO XL99 cell, aCHO-T cell (see e.g. Morgan, D., et al., Biochemistry 26 (1987)2959-2963), or a CHO-S cell, or a Super-CHO cell (Pak, S. C. O., et al.Cytotechnology. 22 (1996) 139-146). If these cells are not adapted togrowth in serum-free medium or in suspension an adaptation prior to theuse in the current method is to be performed. As used herein, theexpression “cell” includes the subject cell and its progeny. Thus, thewords “transformant” and “transformed cell” include the primary subjectcell and cultures derived there from without regard for the number oftransfers or subcultivations. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Variant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included.

The term “expression” as used herein refers to transcription and/ortranslation processes occurring within a cell. The level oftranscription of a nucleic acid sequence of interest in a cell can bedetermined on the basis of the amount of corresponding mRNA that ispresent in the cell. For example, mRNA transcribed from a sequence ofinterest can be quantitated by RT-PCR or by Northern hybridization (seeSambrook, et al., 1989, supra). Polypeptides encoded by a nucleic acidof interest can be quantitated by various methods, e.g. by ELISA, byassaying for the biological activity of the polypeptide, or by employingassays that are independent of such activity, such as Western blottingor radioimmunoassay, using immunoglobulins that recognize and bind tothe polypeptide (see Sambrook, et al., 1989, supra).

An “expression cassette” refers to a construct that contains thenecessary regulatory elements, such as promoter and polyadenylationsite, for expression of at least the contained nucleic acid in a cell.

A “transfection vector” is a nucleic acid (also denoted as nucleic acidmolecule) providing all required elements for the expression of the inthe transfection vector comprised coding nucleic acids/structuralgene(s) in a host cell. A transfection vector comprises a prokaryoticplasmid propagation unit, e.g. for E. coli, in turn comprising aprokaryotic origin of replication, and a nucleic acid conferringresistance to a prokaryotic selection agent, further comprises thetransfection vector one or more nucleic acid(s) conferring resistance toan eukaryotic selection agent, and one or more nucleic acid encoding apolypeptide of interest. Preferably are the nucleic acids conferringresistance to a selection agent and the nucleic acid(s) encoding apolypeptide of interest placed each within an expression cassette,whereby each expression cassette comprises a promoter, a coding nucleicacid, and a transcription terminator including a polyadenylation signal.Gene expression is usually placed under the control of a promoter, andsuch a structural gene is said to be “operably linked to” the promoter.Similarly, a regulatory element and a core promoter are operably linkedif the regulatory element modulates the activity of the core promoter.

A “promoter” refers to a polynucleotide sequence that controlstranscription of a gene/structural gene or nucleic acid sequence towhich it is operably linked. A promoter includes signals for RNApolymerase binding and transcription initiation. The promoter(s) usedwill be functional in the cell type of the host cell in which expressionof the selected sequence is contemplated. A large number of promotersincluding constitutive, inducible and repressible promoters from avariety of different sources, are well known in the art (and identifiedin databases such as GenBank) and are available as or within clonedpolynucleotides (from, e.g., depositories such as ATCC as well as othercommercial or individual sources). A “promoter” comprises a nucleotidesequence that directs the transcription of an operably linked structuralgene. Typically, a promoter is located in the 5′ non-coding oruntranslated region of a gene, proximal to the transcriptional startsite of a structural gene. Sequence elements within promoters thatfunction in the initiation of transcription are often characterized byconsensus nucleotide sequences. These promoter elements include RNApolymerase binding sites, TATA sequences, CAAT sequences,differentiation-specific elements (DSEs; McGehee, R. E., et al., Mol.Endocrinol. 7 (1993) 551-560), cyclic AMP response elements (CREs),serum response elements (SREs; Treisman, R., Seminars in Cancer Biol. 1(1990) 47-58), glucocorticoid response elements (GREs), and bindingsites for other transcription factors, such as CRE/ATF (O'Reilly, M. A.,et al., J. Biol. Chem. 267 (1992) 19938-19943), AP2 (Ye, J., et al., J.Biol. Chem. 269 (1994) 25728-25734), SP1, cAMP response element bindingprotein (CREB; Loeken, M. R., Gene Expr. 3 (1993) 253-264) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre, F. P. and Rousseau, G. G., Biochem. J. 303 (1994) 1-14). Amongthe eukaryotic promoters that have been identified as strong promotersfor high-level expression are the SV40 early promoter, adenovirus majorlate promoter, mouse metallothionein-I promoter, Rous sarcoma virus longterminal repeat, Chinese hamster elongation factor 1 alpha (CHEF-1, seee.g. U.S. Pat. No. 5,888,809), human EF-1 alpha, ubiquitin, and humancytomegalovirus immediate early promoter (CMV IE).

The “promoter” can be constitutive or inducible. An enhancer (i.e., acis-acting DNA element that acts on a promoter to increasetranscription) may be necessary to function in conjunction with thepromoter to increase the level of expression obtained with a promoteralone, and may be included as a transcriptional regulatory element.Often, the polynucleotide segment containing the promoter will includeenhancer sequences as well (e.g., CMV or SV40).

An “enhancer”, as used herein, refers to a polynucleotide sequence thatenhances transcription of a gene or coding sequence to which it isoperably linked. Unlike promoters, enhancers are relatively orientationand position independent and have been found 5′ or 3′ (Lusky, M., etal., Mol. Cell Bio., 3 (1983) 1108-1122) to the transcription unit,within an intron (Banerji, J., et al., Cell, 33 (1983) 729-740) as wellas within the coding sequence itself (Osborne, T. F., et al., Mol. Cell.Bio., 4 (1984) 1293-1305). Therefore, enhancers may be placed upstreamor downstream from the transcription initiation site or at considerabledistances from the promoter, although in practice enhancers may overlapphysically and functionally with promoters. A large number of enhancers,from a variety of different sources are well known in the art (andidentified in databases such as GenBank) and are available as or withincloned polynucleotide sequences (from, e.g., depositories such as theATCC as well as other commercial or individual sources). A number ofpolynucleotides comprising promoter sequences (such as the commonly-usedCMV promoter) also comprise enhancer sequences. For example, all of thestrong promoters listed above may also contain strong enhancers (seee.g. Bendig, M., M., Genetic Engineering 7 (Academic Press, 1988)91-127).

A “nucleic acid conferring resistance to a selection agent” is a nucleicacid that allows cells carrying it to be specifically selected for oragainst, in the presence of a selection agent. Such a nucleic acid isalso denoted as selection marker. Typically, a selection marker willconfer resistance to a selection agent (drug) or compensate for ametabolic or catabolic defect in the host cell. A selection marker canbe positive, negative, or bifunctional. A useful positive selectionmarker is an antibiotic resistance gene. This selection marker allowscells transformed therewith to be positively selected for in thepresence of the corresponding selection agent, i.e. under selectedgrowth in the presence e.g. of the corresponding antibiotic. Anon-transformed cell is not capable to grow or survive under theselective growth conditions, i.e. in the presence of the selectionagent, in culture. Positive selection markers allow selection for cellscarrying the marker, whereas negative selection markers allow cellscarrying the marker to be selectively eliminated. Eukaryotic selectionmarkers include, e.g., the genes for aminoglycoside phosphotransferase(APH) (conferring resistance to the selection agents such as e.g.hygromycin (hyg), neomycin (neomycin phosphotransferase II, neo), andG418), dihydrofolate reductase (DHFR) (conferring resistance to theselection agent methotrexate), thymidine kinase (tk), glutaminesynthetase (GS), asparagine synthetase, tryptophan synthetase(conferring resistance to the selection agent indole), histidinoldehydrogenase (conferring resistance to the selection agent histidinolD), cytidine deaminase, adenosine deaminase and nucleic acids conferringresistance to puromycin, bleomycin, phleomycin, chloramphenicol, Zeocin,and mycophenolic acid. Further selection marker nucleic acids arereported e.g. in WO 92/08796 and WO 94/28143. Prokaryotic selectionmarkers include, e.g. the beta-lactamase gene (conferring resistance tothe selection agent ampicillin).

Expression of a gene is performed either as transient or as permanentexpression. The polypeptide(s) of interest are in general secretedpolypeptides and therefore contain an N-terminal extension (also knownas the signal sequence) which is necessary for the transport/secretionof the polypeptide through the cell wall into the extracellular medium.In general, the signal sequence can be derived from any gene encoding asecreted polypeptide. If a heterologous signal sequence is used, itpreferably is one that is recognized and processed (i.e. cleaved by asignal peptidase) by the host cell. For secretion in yeast for examplethe native signal sequence of a heterologous gene to be expressed may besubstituted by a homologous yeast signal sequence derived from asecreted gene, such as the yeast invertase signal sequence, alpha-factorleader (including Saccharomyces, Kluyveromyces, Pichia, and Hansenulaα-factor leaders, the second described in U.S. Pat. No. 5,010,182), acidphosphatase signal sequence, or the C. albicans glucoamylase signalsequence (EP 0 362 179). In mammalian cell expression the native signalsequence of the protein of interest is satisfactory, although othermammalian signal sequences may be suitable, such as signal sequencesfrom secreted polypeptides of the same or related species, e.g. forimmunoglobulins from human or murine origin, as well as viral secretorysignal sequences, for example, the herpes simplex glycoprotein D signalsequence. The DNA fragment encoding for such a presegment is ligated inframe, i.e. operably linked, to the DNA fragment encoding a polypeptideof interest.

The first aspect of the current invention is a method for therecombinant production of a secreted heterologous immunoglobulin in aCHO cell which comprises:

-   a) providing a CHO cell, which is adapted to growth in suspension    culture, adapted to growth in serum-free medium, and mycoplasma    free;-   b) providing a transfection vector, which comprises the following    elements:    -   a prokaryotic origin of replication,    -   a first nucleic acid sequence conferring resistance to a        prokaryotic selection agent,    -   a second nucleic acid sequence encoding the heavy chain of said        heterologous immunoglobulin and a third nucleic acid sequence        encoding the light chain of said heterologous immunoglobulin,    -   a fourth nucleic acid sequence conferring resistance to a        eukaryotic selection agent,    -   whereby each of said first to fourth nucleic acid sequence is        contained in an expression cassette,-   c) transfecting and selecting said CHO cell, wherein said    transfecting and selecting comprises the following steps in the    following order:    -   (i) transfecting said CHO cell with a transfection vector        comprising said first to third nucleic acid and a fourth nucleic        acid sequence conferring resistance to a first eukaryotic        selection agent,    -   (ii) selecting a CHO cell transfected in (i) by selected growth        in cultivation medium containing said first eukaryotic selection        agent,    -   (iii) transfecting said CHO cell selected in (ii) with a        transfection vector comprising said first to third nucleic acid        and a fourth nucleic acid sequence different from that in the        transfection vector used in (i) conferring resistance to a        second eukaryotic selection agent different to said first        eukaryotic selection agent,    -   (iv) selecting a CHO cell transfected in (iii) by selected        growth in cultivation medium containing said first and said        second eukaryotic selection agent,-   d) cultivating said transfected and selected CHO cell of step c) in    a cultivation medium containing said first and second eukaryotic    selection agent, under conditions suitable for the expression of    said second, and third nucleic acid,-   e) recovering said secreted heterologous immunoglobulin from the    cultivation medium and thereby recombinantly producing a    heterologous immunoglobulin.

The method according to the invention is suited for the production of asecreted heterologous immunoglobulin in large scale, i.e. industrially.The cultivation of a cell for the production of a desired polypeptide inlarge scale generally consists of a sequence of individual cultivations,wherein all cultivations except the final, i.e. the large scale,cultivation, i.e. the last one in the sequence, are performed until acertain cell density is reached in the culture vessel. If thepredetermined cell density is reached the entire cultivation or afraction thereof is used to inoculate the next cultivation vessel, whichhas a larger volume, up to 1000 times the volume of the precedingcultivation. All cultivations which serve as a basis for at least onefurther cultivation in a larger volume are denoted as seed trainfermentations. Only in the large scale cultivation, i.e. in thecultivation which is not intended to serve as the basis for a furthercultivation in a larger volume, which is also denoted as mainfermentation, is the endpoint of the cultivation determined depending onthe concentration of the produced secreted heterologous immunoglobulinin the cultivation medium. The term “large scale” as used within thisapplication denotes the final cultivation of an industrial productionprocess. Preferably a large scale cultivation is performed at a volumeof at least 100 l, more preferably of at least 500 l, most preferably ofat least 1000 l up to a volume of 20,000 l. In one embodiment the final,i.e. large scale, cultivation medium does not contain a eukaryoticselection agent.

In one embodiment the cultivation of said transfected CHO cell isperformed in the presence of said eukaryotic selection agent in a volumeof less than 500 liter and the cultivation of said transfected CHO cellis performed in the absence of said eukaryotic selection agents in avolume of 500 liter or more and that said recovering said secretedheterologous immunoglobulin is from the cultivation medium without saideukaryotic selection agents. In a further embodiment the cultivation iscomprising sequential cultivations with increasing cultivation volume upto a final cultivation volume, whereby the cultivations are performed inthe presence of said eukaryotic selection agents up to a cultivationvolume of 1% (v/v) of the cultivation volume of the final or maincultivation and in the absence of all of said eukaryotic selectionagents in a cultivation volume of more than 1% (v/v) of the cultivationvolume of the final cultivation. In a further embodiment saidcultivation is comprising sequential seed train cultivations withincreasing cultivation volume, whereby each of the seed traincultivations is performed in the presence of said eukaryotic selectionagents and the main fermentation is performed in the absence of all ofsaid eukaryotic selection agents. In one embodiment the cultivation ofsaid transfected CHO cell is performed in the presence of saideukaryotic selection agent in the seed train fermentations and thecultivation of said transfected CHO cell is performed in the absence ofsaid eukaryotic selection agents in the main fermentation and that saidrecovering said secreted heterologous immunoglobulin is from the maincultivation medium not containing said eukaryotic selection agents. Inthese embodiments the eukaryotic selection agents are added during thegrowth phase and omitted during the production phase of said CHO cell.The term “production phase” denotes the cultivation of a CHO cell in alarge volume, i.e. the main fermentation, after which the producedheterologous immunoglobulin is recovered.

In another embodiment of the method according to the invention theproductivity of said CHO cell is over 40 generations not less than 70%and not more than 130% of the productivity after 10 generations ofcultivation as split-batch cultivation. In an embodiment theproductivity of said CHO cells is over 60 generations not less than 50%and not more than 150% of the productivity after 10 generations ofcultivation as split-batch cultivation. The productivity of said CHOcell is at least 1.5 g/l of said heterologous immunoglobulin within 21days as fed-batch cultivation in another embodiment. In one embodimentthe specific productivity of the CHO cell obtained with the methodaccording to the invention is more than 1 μg/10⁶ cells/d, more than 5μg/10⁶ cells/d, or more than 10 μg/10⁶ cells/d. In one embodiment thesecreted heterologous immunoglobulin is a completely processed secretedheterologous immunoglobulin. The term “completely processed secretedheterologous immunoglobulin” denotes an immunoglobulin i) which issecreted to the cultivation medium and whose signal sequences has beencleaved, ii) which comprises an antigen binding region, iii) which hassecondary modifications, such as attached saccharides orpolysaccharides, and/or correctly formed disulfide bonds.

In one embodiment of the invention the heterologous immunoglobulin is ananti-Aβ antibody. In another embodiment the heavy chain variable domainof said anti-Aβ antibody comprises a CDR3 with an amino acid sequenceselected from SEQ ID NO: 1, 2, or 3. In a further embodiment the lightchain variable domain of said anti-Aβ antibody comprises a CDR3 with anamino acid sequence selected from SEQ ID NO: 4, 5, or 6. In a furtherembodiment said anti-Aβ antibody comprises a heavy chain variable domainwith an amino acid sequence selected from SEQ ID NO: 7, 8, or 9. Instill a further embodiment said anti-Aβ antibody comprises a light chainvariable domain with an amino acid sequence selected from SEQ ID NO: 10,11, or 12.

In one embodiment of the invention the heterologous immunoglobulin is ananti-P-Selectin antibody. In a further embodiment said anti-P-Selectinantibody comprises a heavy chain variable domain with an amino acidsequence selected from SEQ ID NO: 13, 14, or 15. In still a furtherembodiment said anti-P-Selectin antibody comprises a light chainvariable domain with an amino acid sequence selected from SEQ ID NO: 16,17, or 18.

In one embodiment of the invention the heterologous immunoglobulin is ananti-IL-13Rα antibody. In a further embodiment said anti-IL-13Rαantibody comprises a heavy chain variable domain with an amino acidsequence selected from SEQ ID NO: 19, 20, 21, 22, or 23. In still afurther embodiment said anti-IL-13Rα antibody comprises a light chainvariable domain with an amino acid sequence selected from SEQ ID NO: 24,25, 26, 27, or 28.

In one embodiment of the invention the heterologous immunoglobulin is ananti-CD4 antibody-conjugate. In another embodiment the heavy chainvariable domain of said anti-CD4 antibody in said conjugate comprises aCDR3 with an amino acid sequence selected from SEQ ID NO: 29, 30, or 31.In a further embodiment the light chain variable domain of said anti-CD4antibody in said conjugate comprises a CDR3 with an amino acid sequenceselected from SEQ ID NO: 32, 33, or 34. In a further embodiment saidanti-CD4 antibody in said conjugate comprises a heavy chain variabledomain with an amino acid sequence selected from SEQ ID NO: 35, 36, or37. In still a further embodiment said anti-CD4 antibody in saidconjugate comprises a light chain variable domain with an amino acidsequence selected from SEQ ID NO: 38, 39, or 40.

A mammalian cell usable for the large scale production of therapeutics,i.e. polypeptides intended for the use in humans, has to fulfilldistinct criteria. Amongst others are these that it has to becultivatable in serum-free, preferably in non-defined mammal-derivedcomponents free medium, or in a serum-free medium supplemented withdefined mammal-derived components. Serum is a mixture of multitude ofcompounds. Normally bovine serum has been used for the cultivation ofmammalian cells. With the arising problem of transmissible diseases fromone species to another the use of serum and other non-definedmammal-derived compounds has to be avoided. The term “non-definedmammal-derived compound” as used within this application denotescompounds which are derived from a mammal, especially preferred from acow, a pig, a sheep, or a lamb, and whose composition can be specifiedto less than 80%, preferably to less than 90% (w/w). A “definedmammal-derived compound” is a compound that is obtained from a mammal,especially preferred from a cow, a pig, a sheep, or a lamb, and whosecomposition can be specified to more than 95% (w/w), preferably to morethan 98% (w/w), most preferably to more then 99% (w/w). An example of adefined mammal-derived compound is cholesterol from ovine wool, andgalactose from bovine milk. In one embodiment the medium can besupplemented with defined or non-defined not mammal-derived compounds.An example of such a not mammal-derived compound is cod-liver oil.

Therefore in one embodiment of the current invention the medium used inthe cultivation is a serum-free medium, or a serum-free mediumsupplemented with defined mammal-derived components, or anmammal-derived component free medium, or a protein-free medium, aprotein-free medium supplemented with defined mammal-derived components,or a chemically defined medium, or a mammal-derived component freemedium, or a defined protein-free medium. Examples of an mammal-derivedcomponent free medium are the CD CHO medium available from InvitrogenCorp., or the ProCHO4 available from Gibco. An example of a protein freemedium is HyQ SFM4CHO available from Hyclone.

In another embodiment of the method according to the invention is themethod beginning with the first transfection and ending with therecovery of the secreted heterologous immunoglobulin performed in thesame medium. The term “in the same medium” denotes within the currentapplication that beginning with the first transfection and ending withthe recovery of the secreted heterologous immunoglobulin from thecultivation medium the same medium is used. This does not denote thatthe same additives have to be added to the medium in all steps, i.e. themedium may be supplemented with different additive in different steps ofthe method. Additives are compounds that are added to a medium in totalto less than 20% (w/w), in one embodiment to less than 15% (w/w), inanother embodiment to less than 10% (w/w). In one embodiment the mediumused in the method according to the invention is the same medium in allsteps and is a medium suitable for the large scale production of thesecreted heterologous immunoglobulin.

It has surprisingly been found that with the method according to theinvention a multiple transfected CHO cell can be obtained that hassimilar growth characteristics and an improved productivity compared toa one-time transfected CHO cell. The term “similar growthcharacteristics” denotes that the multiple transfected CHO cell grows toat least 50% of the cell density within the same time as the one-timetransfected CHO cell. In another embodiment said multiple transfectedCHO cell grows to at least 90% of the cell density as the one-timetransfected cell. In still a further embodiment is the doubling time ofthe multiple transfected cell at most 150% of that of the one-timetransfected cell. In one embodiment said multiple transfected CHO cellis a CHO cell transfected two or three times. In another embodiment themultiple transfected cell has an improved volumetric yield in acultivation medium. The overall productivity of a large scalefermentation process is best determined by the volumetric yield, i.e.the amount of polypeptide per unit volume of the cultivation. Thisvolumetric yield is the product of cell density, specific productivityof each cell and cultivation time. Thus, a cultivation with low celldensity but high specific productivity will have the same volumetricyield in the same time as a cultivation with high cell density but lowspecific productivity in the same cultivation time. Thus, with themultiple transfected CHO cell and the method according to the inventiona CHO cell is obtainable with similar growth characteristics but animproved volumetric yield/productivity compared to one-time transfectedCHO cells.

The secreted heterologous immunoglobulin can be recovered from thecultivation medium with chromatographic methods known to a person ofskill in the art. Therefore in one embodiment the method according tothe invention comprises the final step of purifying said heterologousimmunoglobulin with one or more chromatographic steps.

A vector suited for use in the method according to the inventioncomprises a prokaryotic origin of replication, and a first nucleic acidconferring resistance to a prokaryotic selection agent, and/or a secondnucleic acid encoding the heavy chain of said heterologousimmunoglobulin, and/or a third nucleic acid encoding the light chain ofsaid heterologous immunoglobulin, and a fourth nucleic acid conferringresistance to a eukaryotic selection agent.

The comprised first nucleic acid confers resistance to the addition of aprokaryotic selection agent to the cultivation medium. Exemplaryprokaryotic selection agents are e.g. ampicillin, kanamycin,chloramphenicol, tetracycline, or erythromycin. The term “a nucleic acidconferring resistance to a selection agent” and grammatical equivalentsthereof denotes within the current application that the polypeptideencoded by said nucleic acid can neutralize said selection agent bymodification or degradation or can counteract the effect of saidselection agent. Thus, a cell comprising a nucleic acid conferringresistance to a selection agent has the ability to survive andproliferate with the selection agent present in the cultivation medium.Exemplary eukaryotic selection agents are e.g. neomycin, hygromycin,puromycin, methotrexate, Geneticin® (G418), or mycophenolic acid. Theselection agent is chosen with the proviso that the prokaryotic and theeukaryotic selection agent is not a metal.

The transfection of the provided CHO cell according to the methodaccording to the invention is performed as sequential steps oftransfection and selection. CHO cells suitable in the method accordingto the invention are e.g. a CHO K1 cell, or a CHO DG44 cell, or a CHOXL99 cell, or a CHO DXB11 cell, or a CHO DP12 cell, or a super-CHO cell.Within the scope of the present invention, transfected cells may beobtained with substantially any kind of transfection method known in theart. For example, the nucleic acid may be introduced into the cells bymeans of electroporation or microinjection. Alternatively, lipofectionreagents such as FuGENE 6 (Roche Diagnostics GmbH, Germany), X-tremeGENE(Roche Diagnostics GmbH, Germany), LipofectAmine (Invitrogen Corp.,USA), and nucleotransfection (AMAX Corp.) may be used. Stillalternatively, the nucleic acid may be introduced into the cell byappropriate viral vector systems based on retroviruses, lentiviruses,adenoviruses, or adeno-associated viruses (Singer, O., Proc. Natl. Acad.Sci. USA 101 (2004) 5313-5314).

After the transfection positive transfected cells are selected in thepresence of selection agents, i.e. by selected growth. It hassurprisingly been found that more than one eukaryotic selection agentcan be present in the cultivation medium not interfering with growth andheterologous polypeptide expression if the cultivated CHO cell has beentransfected with all required corresponding nucleic acids conferringresistance to these eukaryotic selection agents according to the currentinvention. It has also been found that CHO cells can be cultivated inthe concomitant presence of three eukaryotic selection agents without areduction of the doubling time to more than 150% of the doubling time ofthe non-transfected or one-time transfected CHO cell. Therefore, themultiple transfected CHO cell comprises nucleic acids, which are in eachtransfection step of the method according to the invention comprising adifferent, not previously transfected, nucleic acid as fourth nucleicacid which confers a new resistance not already present in said CHO cellto a different eukaryotic selection agent. Therefore, after the secondtransfection step a successfully transfected cell is selected for bycultivation in the concomitant presence of two different eukaryoticselection agents. After the third transfection the transfected cell canbe cultivated for selection in the concomitant presence of threedifferent eukaryotic selection agents.

Thus, the vector employed in the different transfection steps accordingto the method according to the invention is at least 95% identical onthe nucleic acid level except for the nucleic acid conferring resistanceto a eukaryotic selection agent, i.e. the fourth nucleic acid.

For the expression of a secreted heterologous immunoglobulin the vectorwith which the CHO cell is transfected and which comprises a nucleicacid conferring resistance to a eukaryotic selection agent alsocomprises a nucleic acid encoding the light chain of said heterologousimmunoglobulin and/or a nucleic acid encoding the heavy chain of saidheterologous immunoglobulin. If the vector comprises only a nucleic acidencoding either the light chain of said immunoglobulin or the heavychain of said immunoglobulin said CHO cell is also transfected in eachstep by another vector comprising a nucleic acid encoding thecorresponding other chain of said immunoglobulin.

In one embodiment the first to fourth nucleic acid sequence comprised inthe transfection vectors according to the invention (i.e. the first,second, and third transfection vector) is contained in an expressioncassette. An “expression cassette” refers to a construct that containsthe necessary regulatory elements, such as promoter and polyadenylationsite, for expression of at least the contained nucleic acid in a cell,e.g. a promoter, a nucleic acid to be expressed, and a transcriptionterminator including a polyadenylation signal. The promoter contained inthe expression cassette determines the amount of transcription of theoperably linked nucleic acid and therewith it determines the amount ofthe translation of said nucleic acid. A first promoter inducing a largeramount of translation of a nucleic acid compared to a second promoter istermed a “stronger promoter” with respect to said second promoter. It isintended to produce the secreted heterologous immunoglobulin and not thepolypeptide conferring resistance to a selection agent. Thus, thecapacity of the host cells transcription and translation machinery hasto be split up correspondingly. Therefore, in one embodiment thepromoter employed for the transcription of said second and third nucleicacids is different from the promoter employed for the transcription ofsaid fourth nucleic acid. In another embodiment is the amount oftranscript of said second and third nucleic acid encoding the chains ofsaid heterologous immunoglobulin larger than the amount of transcript ofsaid forth nucleic acid conferring resistance to a selection agent.Thus, the promoter employed for the expression of said second and thirdnucleic acid is stronger than the promoter employed for the expressionof said fourth nucleic acid. In another embodiment is the promoteremployed for the transcription of said second and third nucleic acidsthe same but different from the promoter of said fourth nucleic acid. Inone embodiment the promoter for the expression of said second and thirdnucleic acid is the CMV promoter or a variant thereof and the promoterfor the expression of said fourth nucleic acid is the SV40 promoter or avariant thereof.

In a further embodiment of the method according to the invention thecodon usage of said second and third nucleic acid is optimized for theexpression in CHO cells. This allows a more efficient use of thetransfer-RNAs present in the recombinant CHO cell. In another embodimentsaid second and/or third nucleic acid comprise an intronic nucleic acidsequence, in another embodiment the intronic nucleic acid is amouse/human hybrid intron. In the genome of eukaryotic cells the genomicDNA sequences contain coding (exonic) and non-coding (intronic) nucleicacid sequences. After transcription of the DNA to the pre-mRNA, thepre-mRNA also contains these intronic and exonic nucleic acid sequences.Prior to translation the non-coding intronic nucleic acid sequences areremoved during mRNA processing by splicing them out of the primary mRNAtranscript to generate the mature mRNA. The splicing of the primary mRNAis controlled by a splice donor site in combination with a properlyspaced apart splice acceptor site. The splice donor site is located atthe 5′ end and the splice acceptor site is located at the 3′ end of anintronic sequence and both are only partly removed during the pre-mRNAsplicing.

To produce a secreted polypeptide, the nucleic acid(s) encoding thechains of the heterologous immunoglobulin include a DNA segment thatencodes a signal sequence/leader peptide. The signal sequence directsthe newly synthesized polypeptide to and through the Endoplasmaticreticulum (ER) membrane where the polypeptide can be routed forsecretion. The signal sequence is cleaved off by a signal peptidasesduring crossing of the ER membrane. As for the function of the signalsequence the recognition by the host cell's secretion machinery isessential. Therefore, the used signal sequence has to be recognized bythe host cell's proteins and enzymes of the secretion machinery.

In one embodiment the method according to the invention comprises athird transfection step in step c):

-   -   (v) transfecting said CHO cell selected in (iv) with said vector        comprising a fourth nucleic acid sequence different from that in        the transfection vector used in (i) and (iii) conferring        resistance to a third eukaryotic selection agent, which is        different from said first and said second eukaryotic selection        agent,    -   (vi) selecting a CHO cell transfected in (v) by selected growth        in a cultivation medium containing said first and said second        and said third eukaryotic selection agent.

In this embodiment the cultivation medium employed for the cultivationof said transfected CHO cell in step d) further comprises a thirdeukaryotic selection agent.

A second aspect of the current invention is a CHO cell expressing asecreted heterologous immunoglobulin obtainable with the followingmethod:

-   a) providing a CHO cell, which is    -   adapted to growth in suspension culture,    -   adapted to growth in serum-free medium,    -   mycoplasma free,-   b) providing a nucleic acid comprising    -   a prokaryotic origin of replication,    -   a first nucleic acid sequence conferring resistance to a        prokaryotic selection agent,    -   a second nucleic acid sequence encoding the heavy chain of said        heterologous immunoglobulin, and a third nucleic acid sequence        encoding the light chain of said heterologous immunoglobulin,    -   whereby a first transfection vector is provided which comprises        said provided nucleic acid and an additional fourth nucleic acid        sequence conferring resistance to a first eukaryotic selection        agent,    -   whereby a second transfection vector is provided which comprises        said provided nucleic acid and an additional fourth nucleic acid        sequence different from the fourth nucleic acid in said first        transfection vector conferring resistance to a second eukaryotic        selection agent, whereby said second eukaryotic selection agent        is different to said first eukaryotic selection agent,-   c) transfecting and selecting said CHO cell, wherein said    transfecting and selecting comprises the following steps in the    following order:    -   (i) transfecting said CHO cell with said first transfection        vector,    -   (ii) selecting a CHO cell transfected in (i) by selected growth        in a cultivation medium containing a first eukaryotic selection        agent to which the first transfection vector confers resistance,    -   (iii) transfecting said CHO cell selected in (ii) with said        second transfection vector,    -   (iv) selecting a CHO cell transfected in (iii) by selected        growth in a cultivation medium containing said first eukaryotic        selection agent, to which the first transfection vector confers        resistance, and said second eukaryotic selection agent, to which        the second transfection vector confers resistance.

The term “virus free” which is used within this application denotes thatthe CHO cell does not contain any viral nucleic acid which would resultif expressed during cultivation in harmful, in down stream processingoperations not separatable products for humans.

The following examples, and figures are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Annotated plasmid map of plasmid p5128.

FIG. 2 Annotated plasmid map of plasmid p5137.

FIG. 3 Annotated plasmid map of plasmid p5151.

FIG. 4 Annotated plasmid map of plasmid p5057.

FIG. 5 Annotated plasmid map of plasmid p5069.

FIG. 6 (A) Antibody titers of clones obtained after subcloning withlimited dilution and of clones obtained with the method according to theinvention; X-axis: (1) G24, (2) limited dilution, (3) method accordingto the invention; Y-axis: immunoglobulin concentration [μg/ml].

-   -   (B) Specific production rates of clones obtained after        subcloning with limited dilution and of clones obtained with the        method according to the invention; X-axis: (1) G24, (2) limited        dilution, (3) method according to the invention; Y-axis:        specific production rate [pg/d*cell].

FIG. 7 SDS-Page after protein-A HPLC purification of the antibody. Forthe four samples 35-45, 37-65, 39-4 and 43-16 two bands are visible, theupper being the heavy chain, the lower being the light chain. Sample25g7 is a control antibody with antibody-related side products (abovethe heavy chain and between heavy and light chain). Samples: (1)Molecular weight marker, (2) 35-45, (3) 37-65, (4) 39-4, (5) 43-16), (6)25g7, (7) Reference antibody, (8) Medium 25×.

FIG. 8 Annotated plasmid map of plasmid p6311.

FIG. 9 Annotated plasmid map of plasmid p5321.

EXAMPLES

Materials & Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered according to EU numbering(Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85;Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md., (1991)).

Recombinant DNA Techniques:

Standard methods were used to manipulate DNA as described in Sambrook,J., et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis:

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 100-600 bp long gene segments, which are flankedby singular restriction endonuclease cleavage sites, were assembled byannealing and ligation of oligonucleotides including PCR amplificationand subsequently cloned into the pCR2.1-TOPO-TA cloning vector(Invitrogen Corp., USA) via A-overhangs or pPCR-Script Amp SK(+) cloningvector (Stratagene Corp., USA). The DNA sequence of the subcloned genefragments were confirmed by DNA sequencing.

Protein Determination:

Protein concentration was determined by determining the optical density(OD) at 280 nm, using the molar extinction coefficient calculated on thebasis of the amino acid sequence.

Antibody Titer Determination:

Antibody titers were determined either by anti-human Fc ELISA or byProtein A chromatography using the autologous purified antibody as areference.

SDS-PAGE

LDS sample buffer, fourfold concentrate (4×): 4 g glycerol, 0.682 gTRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS (lithium dodecylsulfate), 0.006 g EDTA (ethylene diamin tetra acid), 0.75 ml of a 1% byweight (w/w) solution of Serva Blue G250 in water, 0.75 ml of a 1% byweight (w/w) solution of phenol red, add water to make a total volume of10 ml.

The culture broth containing the secreted antibody was centrifuged toremove cells and cell debris. An aliquot of the clarified supernatantwas admixed with ¼ volumes (v/v) of 4xLDS sample buffer and 1/10 volume(v/v) of 0.5 M 1,4-dithiotreitol (DTT). Then the samples were incubatedfor 10 min. at 70° C. and protein separated by SDS-PAGE. The NuPAGE®Pre-Cast gel system (Invitrogen Corp.) was used according to themanufacturer's instruction. In particular, 10% NuPAGE® Novex® Bis-TRISPre-Cast gels (pH 6.4) and a NuPAGE® MOPS running buffer was used.

Western Blot

Transfer buffer: 39 mM glycine, 48 mM TRIS-hydrochloride, 0.04% byweight (w/w) SDS, and 20% by volume methanol (v/v)

After SDS-PAGE the separated antibody chains were transferredelectrophoretically to a nitrocellulose filter membrane (pore size: 0.45μm) according to the “Semidry-Blotting-Method” of Burnette (Burnette, W.N., Anal. Biochem. 112 (1981) 195-203).

Example 1

Expression Vector for Expressing an Anti-Aβ Antibody

An example (preferably monoclonal) antibody for which a cell line forexpression can be obtained according to the current invention is anantibody against the amyloid β-A4 peptide (anti-Aβ antibody). Such anantibody and the corresponding nucleic acid sequences are, for example,reported in WO 2003/070760 or US 2005/0169925 or in SEQ ID NO: 1 to 12.

The anti-Aβ antibody expressing Chinese hamster ovary (CHO) cell linewas generated by three successive complete transfections and selectioncampaigns.

A genomic human κ-light chain constant region gene segment (C-kappa,C_(L)) was added to the light chain variable region of the anti-Aβantibody, while a human γ1-heavy chain constant region gene segment(C_(H1)-Hinge-C_(H2)-C_(H3)) was added to the heavy chain variableregion of the anti-Aβ antibody. The complete κ-light and γ1-heavy chainantibody genes were then joined with a human cytomegalovirus (HCMV)promoter at the 5′-end and a human immunoglobulin polyadenylation signalsequence at the 3′-end.

a) Heavy Chain Expression Cassette

The transcription unit of the anti-Aβ antibody heavy chain is composedof the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   a 5′-untranslated region derived from a human antibody germline        gene,    -   the anti-Aβ antibody heavy chain variable domain including a        signal sequence derived from a human antibody germline gene,    -   a human/mouse heavy chain hybrid intron 2 including the mouse Ig        heavy chain enhancer element (see e.g. (Neuberger, M. S.,        EMBO J. 2 (1983) 1373-1378),    -   the genomic human γ1-heavy chain gene constant region,    -   the human immunoglobulin γ1-heavy chain polyadenylation (“poly        A”) signal sequence,    -   the unique restriction sites AscI and SgrAI at the 5′- and        3′-end, respectively.        b) Light Chain Expression Cassette

The transcription unit of the anti-Aβ antibody light chain is composedof the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   a 5′-untranslated region derived from a human antibody germline        gene,    -   the anti-Aβ antibody light chain variable region including a        signal sequence derived from a human antibody germline gene,    -   a human/mouse κ-light gene hybrid intron 2 including the mouse        Ig χ-light chain enhancer element (Picard and Schaffner, A        lymphocyte-specific enhancer in the mouse immunoglobulin kappa        gene. Nature 307 (1984) 80-82),    -   the human κ-light gene constant region (C-kappa),    -   the human immunoglobulin κ-polyadenylation (“poly A”) signal        sequence,    -   the unique restriction sites Sse8387 and FseI at the 5′- and        3′-end, respectively.        c) Expression Plasmids 5128, 5137, and 5151

For expression and production of the anti-Aβ antibody the light andheavy chain expression cassettes were placed on a single expressionvector (heavy chain upstream of light chain in clockwise orientation).Three identical expression vectors were generated differing only in theselectable marker gene included, in particular, in the gene conferringresistance to the selection agent neomycin, hygromycin, or puromycin.The vectors also include a mouse DHFR gene which was not used forselection or amplification.

The expression vectors contain beside the light and heavy chainexpression cassette the following elements:

-   -   a selectable marker (either a neomycin, hygromycin or puromycin        resistance gene),    -   an origin of replication allowing for the replication of the        plasmid in E. coli,    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli,    -   a mouse derived DHFR gene.

The plasmid map of the expression vector 5128 containing a hygromycinselectable marker gene is shown in FIG. 1. The plasmid map of theexpression vector 5137 containing a neomycin selectable marker gene isshown in FIG. 2. The plasmid map of the expression vector 5151containing a puromycin selectable marker gene is shown in FIG. 3.

Example 2

Transfection and Selection of a CHO Cell Expressing an Anti-Aβ Antibody

Parent CHO-K1 cells, pre-adapted to growth in serum-free suspensionculture in synthetic animal component free ProCHO4 medium (CambrexCorp.) containing 8 mM glutamine and 1×HT supplement (Gibco/Invitrogen)were used as host cell line. This supplemented ProCHO4 medium isdesignated in the following as ProCHO4-complete medium. The adherentgrowing CHO-K1 parent cell line was received from ATTC as ATCC CCL-61.

The preadapted parent host cells were propagated in suspension insynthetic, animal component-free ProCHO4-complete medium under standardhumidified conditions (95%, 37° C., and 5% CO₂). On regular intervalsdepending on the cell density the cells were splitted into fresh medium.The cells were harvested by centrifugation in the exponential growthphase, washed once in sterile Phosphate Buffered Saline (PBS) andresuspended in sterile PBS.

Prior to transfection the anti-Aβ antibody expressing plasmids werelinearized within the β-lactamase gene (E. coli ampicillin resistancemarker gene) using the restriction endonuclease enzyme PvuI or AviII.The cleaved DNA was precipitated with ethanol, dried under vacuum, anddissolved in sterile PBS.

In general, for transfection, the (parent or already transfected) CHOcells were electroporated with 20-50 μg linearized plasmid DNA perapproximately 10⁷ cells in PBS at room temperature. The electroporationswere performed with a Gene Pulser XCell electroporation device (Bio-RadLaboratories) in a 2 mm gap cuvette, using a square wave protocol with asingle 180 V pulse. After transfection, the cells were plated out inProCHO4-complete medium in 96-well culture plates. After 24 h of growtha solution containing one or more selection agents were added(ProCHO4-complete selection medium; G418: 400 μg/ml; hygromycin: 600μg/ml; puromycin: 8 μg/ml). Once a week the ProCHO4-complete selectionmedium was replaced. The antibody concentration of the anti-Aβ antibodywas analyzed with an ELISA assay specific for human IgG1 in the culturesupernatants.

For selection of high-yield anti-Aβ antibody production cell lines theproductivity was tested in ProCHO4-complete selection medium afterpropagation in 6-well culture plates, T-flasks and/or Erlenmeyer shakeflasks using an anti-human IgG1 ELISA and/or analytic Protein A HPLC.

Subclones were obtained by two methods, Limiting Dilution (LD) andFluorescence Activated Cell Sorting (FACS).

Limiting Dilution:

For limiting dilution cells were plated out in ProCHO4-conditionedmedium (consisting of 50% (v/v) fresh ProCHO4-complete selection mediumand 50% (v/v) ProCHO4-complete conditioned selection medium derived fromthe cells to be propagated) at a cell density of 0.5-2 cells per 0.1 mlmedium per well of a 96-well culture plate. Once a week the medium wasreplaced by ProCHO4-complete selection medium. The antibodyconcentration of the anti-Aβ antibody was analyzed by an ELISA assayspecific for human IgG1 in the culture supernatants.

Single Cell Deposition by Flow Cytometry Including Identification andIsolation of Clones:

The identification and isolation of stably transfected clones wasperformed with the aid of a cell surface labeling technique usingfluorescently tagged Protein A that binds to secreted but stillmembrane-attached antibodies. The fluorescence intensity of the stainedcells was used as criterion for cell selection.

In the case of fluorescence activated cell sorting the electroporatedpopulation of cells were directly seeded into T-flasks inProCHO4-complete medium. The appropriate selection agent or agents(G418, hygromycin, and/or puromycin) was/were added to the culture oneday after transfection and the transfectant pool was expanded.

Cells from the expanded transfectant pool were first treated withAccumax (PAA Laboratories) for 15 minutes at 37° C. and then passedthrough a 40 μM nylon mesh to remove remaining large cell aggregates.The cells were collected by centrifugation, resuspended in PBScontaining 5% FCS (Gibco/Invitrogen) at a cell density of 10⁶ to 10⁷cells/ml and incubated for 20 minutes on ice. Thereafter, the cells werestained with 10 ng/ml Protein A Alexa Fluor 488 (Molecular Probes Inc.)in a volume of 8 ml FCS-PBS for 30 minutes on ice in the dark.Afterwards, the cells were washed once with 5% FCS-PBS and once withProCHO4 medium containing 8 mM Ultra Glutamine (Cambrex Corp.), 1×HTsupplement and 5% FCS. Finally the cells were resuspended in thesupplemented ProCHO medium used for washing at a cell density of 10⁶ to10⁷ cells/ml and transferred to a BD FACSAria cell sorter (BDBiosciences).

Single cells were sorted by flow cytometry and deposited in wells of96-well culture plates containing of ProCHO4-conditioned medium. Theselected and deposited cells encompassed cells with the top 10%, 7%, or4% of fluorescence intensity of the gated live cells. After 48 hoursProCHO4 complete selection medium containing the appropriate selectionagent in 2-fold concentration was added to each well. Once a week themedium was replaced with ProCHO4-complete selection medium. The antibodyconcentration of the anti-Aβ antibody was analyzed with an ELISA assayspecific for human IgG1 in the culture supernatants.

Transfection and Selection Steps:

For the first transfection and selection step the plasmid 5137 has beenused. Plasmid 5137 has been transfected with electroporation into parentcell line adapted to growth in ProCHO4-complete medium. The transfectedcells were cultivated in ProCHO4-complete medium supplemented with up to700 μg/ml G418 in 96 well plates. The antibody concentration in theculture supernatants was evaluated by an anti-human IgG1 ELISA.Approximately 1000 clones have been tested and the selected of them werefurther cultivated in 24-well plates, 6-well plates and subsequently inshaker flasks. The growth and productivity of approximately 20 cloneswas assessed in static and suspension cultures by anti-human IgG1 ELISAand/or analytic protein A HPLC. The best clone (best clone does notdenote the most productive clone it denotes the clone with the bestproperties for the further steps) was subcloned by limited dilution inProCHO4-conditioned medium supplemented with 700 μg/ml G418. Theselected clone was named 8C8.

For the second transfection and selection step the plasmid 5128 has beenused. Plasmid 5128 has been transfected with electroporation into cellline clone 8C8 cultivated in ProCHO4-complete medium supplemented with700 μg/ml G418. The transfected cells were expanded for about two tothree weeks in ProCHO4-conditioned medium supplemented with 200 μg/mlG418 and 300 μg/ml hygromycin (ProCHO4-double selection medium). Singleantibody secreting cells were identified and deposited on the basis oftheir fluorescence intensity after staining with a Protein A Alexa Fluorconjugate by FACS analysis. The deposited cells were cultivated inProCHO4-double selection medium in 96 well plates. The antibodyconcentration in the culture supernatants was evaluated by an anti-humanIgG1 ELISA. Approximately 500 clones have been tested and the selectedof them were further cultivated in 24-well plates, 6-well plates andsubsequently in shaker flasks. The growth and productivity ofapproximately 14 clones was assessed in static and suspension culturesby anti-human IgG1 ELISA and/or analytic Protein A HPLC. The selectedclone was named 4F5.

For the third transfection and selection step the plasmid 5151 has beenused. Plasmid 5151 has been transfected with electroporation into cellline clone 4F5 cultivated in ProCHO4-double selection medium. Thetransfected cells were expanded for about two to three weeks inProCHO4-triple selection medium (ProCHO4-conditioned medium supplementedwith 200 μg/ml G418 and 300 μg/ml hygromycin and 4 μg/ml puromycin).Single antibody secreting cells were identified and deposited on thebasis of their fluorescence intensity after staining with a Protein AAlexa Fluor conjugate by FACS analysis. The deposited cells werecultivated in ProCHO4-triple selection medium in 96 well plates. Theantibody concentration in the culture supernatants was evaluated by ananti-human IgG1 ELISA. Approximately 500 clones have been tested and theselected of them were further cultivated in 24-well plates, 6-wellplates and subsequently in shaker flasks. The growth and productivity ofapproximately 10 clones was assessed in static and suspension culturesby anti-human IgG1 ELISA and/or analytic protein A HPLC. The selectedclone was named 20F2.

Clone 20F2 has been selected based on his growth, productivity, andproduct quality characteristics after growth in fed-batch suspensionculture in ProCHO4-triple selection medium, i.e. in the concomitantpresence of the three selecting agents G418, hygromycin, and puromycin.

Clone Characteristics:

As can be seen from the following table the doubling time and celldensity after three days of cultivation were comparable when the basiccell line CHO-K1 (wild-type) and the selected clones are compared.

TABLE 1 Growth characteristics Doubling Starting Cell density Viabilitytime cell density at day 3 at day 3 Clone [h] [10⁶ cells/ml] [10⁶cells/ml] [%] CHO-K1 22-23 3 18-20 97-98 (wild-type) 8C8 26-28 3 12-1596-98 4F5 22-24 3 24-27 96-97 20F2 24-26 2 23-26 97-98

Example 3

Stability of Clone 20F2 Expressing an Anti-Aβ Antibody

Stability of growth and product formation was evaluated in sequentialcell subculture over a time period of 60 days (about 60 generations) inthe presence and absence of the selection agents (with and withoutantibiotics). The cultivation was performed as described above.

TABLE 2 Characteristics of clone 20F2. Clone 20F2 cultivation in thecultivation in presence of three the absence of Parameter selectionagents selection agents Mean value viability [%] 97 97 Mean valuedoubling time [h] 27 26 Mean value SPR [pg/c/d] 11 9Following extensive passage (up to generation 60) no evidence wasobtained indicating that the anti-Aβ antibody producing clone 20F2 wasunstable with respect to cell growth and product formation in thepresence or absence of the three selection agents, respectively.

Example 4

Expression Vector for Expressing an Anti-P-Selectin Antibody

Another example (preferably monoclonal) antibody for which a cell linefor expression can be obtained according to the current invention is anantibody against the human P-Selectin glycoprotein (anti-P-Selectinantibody). Such an antibody and the corresponding nucleic acid sequencesare for example described in WO 2005/100402, or US 2005/0226876 or SEQID NO: 13 to 18.

The anti-P-Selectin antibody expressing Chinese hamster ovary cell linewas generated by two successive complete transfections and cloneselection campaigns.

A genomic human kappa-light chain constant region gene segment (C-kappa)was added to the light chain variable region of the anti-P-Selectinantibody, whereas a human gamma 4-heavy chain constant region genesegment (C_(H1)-Hinge-C_(H2)-C_(H3)) was added to the heavy chainvariable region of the anti-P-Selectin antibody. The completekappa-light and gamma 4-heavy chain antibody genes were then joined witha human cytomegalovirus immediate early promoter and enhancer (CMV IE)at the 5′-end and the Simian Virus 40 early polyadenylation (SV 40 earlypoly A) signal sequence at the 3′-end.

-   a) Heavy Chain Expression Cassette

The transcription unit of the anti-P-Selectin antibody heavy chain iscomposed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (CMV IE),    -   a 5′-untranslated region (5′ UTR),    -   the coding sequence for the anti-P-Selectin antibody gamma        4-heavy chain including a signal peptide in an intron-exon gene        structure,    -   the SV 40 early poly A signal sequence.

-   b) Light Chain Expression Cassette

The transcription unit of the anti-P-Selectin antibody light chain iscomposed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (CMV IE),    -   a 5′-untranslated region (5′ UTR),    -   the coding sequence for the anti-P-Selectin kappa-light chain in        an intron-exon gene structure,    -   the SV 40 early poly A signal sequence.

-   c) Expression Plasmids 5057 and 5069

For the expression and production of the anti-P-Selectin antibody thelight and heavy chain expression cassettes were placed on a singleexpression vector (light chain upstream of heavy chain). Two identicalexpression vectors were generated differing only in the selectablemarker gene included, in particular, the murine dihydrofolate reductase(DHFR) gene or a neomycin resistance gene.

The expression vectors contain beside the light and heavy chainexpression cassette the following elements:

-   -   a selectable marker, either the murine DHFR gene or a gene        conferring resistance to the selection agent neomycin under the        control of the SV40 early promoter and origin,    -   an origin of replication allowing for the replication of the        plasmid in E. coli taken from pUC19 (pUC origin),    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

The plasmid map of the expression vector 5057 containing the murine DHFRmarker gene is shown in FIG. 4. The plasmid map of the expression vector5069 containing a neomycin selectable marker gene is shown in FIG. 5.

Example 5

Transfection and Selection of a CHO Cell Line Expressing anAnti-P-Selectin Antibody

CHO-K1 cells, pre-adapted to growth in serum-free suspension culture inprotein-free HyQ SFM4CHO medium (Hyclone, Cat. No. SH30549) supplementedwith defined animal-derived components (cholesterol from ovine wool andcod-liver oil) were used as the host cell line. The cells werepropagated in shake flasks in protein-free HyQ SFM4CHO medium understandard humidified conditions (95%, 37° C., and 5% CO₂) and underconstant agitation at 150 rpm/min. Depending on the cell density thecells were split into fresh medium.

The adherent CHO-K1 cell lines had been obtained from the American TypeCulture Collection as ATCC CCL-61.

First Transfection and Selection

Prior to transfection the expression plasmid 5057 was linearized withinthe beta-lactamase gene using the restriction enzyme PvuI. The cleavedDNA was purified using QiaQuick spin columns (Qiagen) according to themanufacturer's recommendations.

Transfection was carried out by electroporation using Gene Pulser XCell(BIO-RAD) and 0.2 cm-cuvettes (BIO-RAD, Cat. No. 165-2086). Fortransfection 10⁶ to 10⁷ CHO-K1 cells were harvested by centrifugation,resuspended in PBS, transferred to the cuvette and mixed with 20-50 μglinearized plasmid DNA. The cells were exposed to a single square wavepulse (160 V, 15 ms) and subsequently diluted in HyQ SFM4CHO medium to adensity of approx. 4×10⁵ cells/ml and seeded in a T75 cell cultureflask. After 48 hours of propagation without the supplementation of aselection agent, the cells were diluted in HyQ SFM4CHO mediumsupplemented with 200 nM MTX to a density of 10⁴ to 10⁵ cells/ml andseeded in 96-well plates with 3-7000 cells per well. After approx. twoweeks, fresh medium was added per well and after additional two weeksthe culture medium was completely replaced by fresh medium. Four dayslater the culture supernatants were tested for antibody production byanti-human Fc ELISA. In total approximately 600 clones were screened.

45 clones with antibody titers of more than 10 μg/ml were picked andtransferred to 48-well plates. The clones were expanded to shaker flasksover additional passages and subsequently transferred to serum freeproduction medium for the final productivity assessment. A 125 ml shakerflask was inoculated with 10⁵ to 10⁶ cells/ml in medium supplementedwith 200 nM MTX. Viable cell density and viability were monitored overone week. Antibody titers were measured by Protein A chromatography onthe final day. Based on these data, clone G24 was selected for furtherdevelopment. G24 reached a maximal viable cell density of 3.3×10⁶cells/ml. The antibody titer was 402 μg/ml. The average specificproduction rate (SPR) was 28 pg/(cell*d).

Second Transfection and Selection:

Clone G24 was subjected to a second transfection. For the secondtransfection plasmid 5069 was used. Linearization and purification ofthe plasmid as well as electroporation of G24 were performed asdescribed for the first transfection. After 48 hours of propagationwithout selection pressure, the cells were diluted in HyQ SFM4CHO mediumsupplemented with 200 nM MTX and 400 μg/ml G418 to a density of 10³ to10⁴ cells/ml and seeded in 96-well plates with 500 cells per well. Afterapprox. two weeks, fresh medium was added per well and after anadditional week the culture medium was completely replaced by freshmedium. Four days later the culture supernatants were tested forantibody production by anti-human Fc ELISA. In total approximately 220clones were screened.

Then 13 clones with antibody titers of more than 150 μg/ml were pickedand transferred to 24-well plates. The clones were expanded to shakerflasks over additional passages and subsequently transferred to serumfree production medium for the final productivity assessment. A shakerflask was inoculated with 10⁵ to 10⁶ cells/ml in 50 ml mediumsupplemented with 200 nM MTX and 400 μg/ml G418. Viable cell density andviability were monitored over one week. Antibody titers were measured byProtein A chromatography on the final day. Based on these data, cloneG24_x6 was considered the best clone. G24_x6 reached a maximal viablecell density of 3.0×10⁶ cells/ml. The antibody titer was 685 μg/ml. Theaverage specific production rate (SPR) from was 48 pg/(cell*d).

Limiting Dilution:

To compare the method according to the invention with simple subcloningwith respect to their effect on productivity we subjected clone G24 tolimited dilution or single cell deposition in 96-well plates.

For limiting dilution the cells were seeded in 96-well plates in HyQSFM4CHO medium supplemented with 50% (v/v) conditioned medium, 10% FCSand 200 nM MTX at 0.5 cells/well. Alternatively 1 cell/well wasdeposited in 96-well plates by FACS. After 10 days, fresh HyQ SFM4CHOmedium, 200 nM MTX without FCS was added per well and after anadditional week the culture medium was completely replaced by HyQSFM4CHO medium, 200 nM MTX. Four days later the culture supernatantswere tested for antibody production by anti-human Fc ELISA. In totalapproximately 230 clones were screened.

Eleven subclones with antibody titers of more than 130 μg/ml weretransferred to 24-well plates. After passages in 6-well plates, theclones were transferred to shaker flasks and subsequently transferred toserum free production medium for the final productivity assessment. Ashaker flask was inoculated with 10⁵ to 10⁶ cells/ml in mediumsupplemented with 200 nM MTX. Viable cell density and viability weremonitored over one week. Antibody titers were measured by Protein Achromatography on the final day. Based on these data G24_(—)13 wasconsidered the best clone. G24_(—)13 reached a maximal viable celldensity of 3.6×10⁶ cells/ml. The antibody titer was 472 μg/ml. Theaverage the specific production rate (SPR) was 31 pg/(cell*d).

Table 3 summarizes the productivity data of best performing subcloneG24_(—)13 and the best performing clone G24_x6 obtained with the methodaccording to the invention in comparison to their parental clone G24.With the method according to the invention a clone with volumetric andspecific productivity increased by more than 50% can be obtained whereasafter subcloning only a minor increase of both parameters was observed.

FIG. 6 shows an overview of the volumetric (A) and specific (B)productivities of all subclones of G24 that had been investigated inshake flasks. As can be seen, the average volumetric and specificproductivity of the clones obtained with the method according to theinvention was significantly higher than after subcloning.

TABLE 3 Productivity of the best producing clones compared to theparental clone G24. G24_x6 (method G24_13 according to G24 (Subclone)the invention) Antibody concentration 402 472 685 in the supernatant[μg/ml] SPR pg/(cell*d)] 28 31 48 Max. cell density 33 36 30 [10⁵/ml]Clone Characteristics:

As can be seen from the following table the doubling time and the celldensity after three days of cultivation were comparable when theone-time transfected cell line G24 and the selected clones are compared.

TABLE 4 Growth characteristics Doubling Starting Cell density Viabilitytime cell density at day 3 at day 3 Clone [h] [10⁶ cells/ml] [10⁶cells/ml] [%] G24 29 0.3 0.7 91 G24_13 27 0.3 2.0 91 G24_x6 24 0.3 2.593

Example 6

Transfection and Selection of a CHO Cell Line Expressing anAnti-P-Selectin Antibody

CHO-DG44 cells pre-adapted to growth in serum-free suspension culture inprotein-free HyQ SFM4CHO medium (Hyclone, Cat. No. SH30549) were used asthe host cell line. The host cell line was cultured in commercial mediumHyQ SFM4CHO-utility (Hyclone, Cat. No. SH30516) during transfections,screening and subcloning steps.

First Transfection and Selection

Prior to transfection the expression plasmid 5057 (FIG. 4) waslinearized within the beta-lactamase gene using the restriction enzymePvuI.

The transfection of the host cell line was performed bynucleotransfection provided by AMAXA (Nucleofector Kit T, Cat. No.VCA-1002, Transfection program U-17). Cells were cultured in mediumsupplemented with 10% fetal calf serum for 48 h after transfection.

Transfected cells were plated on 96-well plates with 1000 cells per wellin medium supplemented with 10% fetal calf serum in the presence of 40nM methotrexate (MTX) as selection agent and incubated for approx. threeweeks.

Antibody concentration was determined by ELISA in the supernatant of the96-well plates. About 400 primary clones were screened. Twenty-fourclones with the highest antibody productivity were transferred to24-well plates and cultivated in the presence of the selection agentwithout supplementation with fetal calf serum. Product quality wasanalyzed by Western Blotting detecting light and heavy antibody chains.Nine clones which showed the highest productivity and which expressedantibody without detectable antibody derived side products (Westernblot) were expanded into shake flasks.

Productivity was analyzed in batch shake flasks after 7 and 10 days ofincubation. Product quality was assessed by SDS-PAGE after Protein-AHPLC purification (FIG. 7). Best product concentration was reached withclone 43-16. Best specific productivity per cell was achieved with clone35-45. Both clones showed no detectable side products in the SDS-PAGE.Both clones were selected for subcloning by limiting dilution.

Parental clones 35-45 and 43-16 were subcloned by limiting dilution on96-well plates in commercial HyQ medium supplemented with 5% (v/v) fetalcalf serum in the presence of 20 nM MTX. After 20 days of incubationantibody production was screened by ELISA. Best subclones in terms ofproductivity were expanded to shake flasks and subsequently transferredto serum free production medium for the final productivity assessment.The two best subclones, 35-45-F2 and 43-16-A10, of the parental clones35-45 and 43-16 were assessed in standard batch shake flask assay.Productivity was 270 μg/ml and 185 μg/ml after 7 days and 337 μg/ml and343 μg/ml after 10 days, respectively.

Second Transfection and Selection:

Subclone 43-16-A10 was transfected with the expression vector p5069(FIG. 5) using the nucleofection method (Amaxa Nucleofector Kit T,VCA-1002, Transfection program U-17). The second transfection was alsocarried out in Hyclone medium: HyQ SFM4CHO-utility (Cat. No. SH30516)supplemented with 10% fetal calf serum and 20 nM MTX. Two days after thesecond transfection cells were transferred to 96-well plates with 1000cells per well. As second selection agent 250 μg/ml G418 was added.

After cultivation for two weeks more than 2000 primary wells werescreened by antibody titer determination by anti-human Fc ELISA. Fiftyclones with highest productivity were transferred into 24-well platesand screened a second time by anti-human Fc ELISA three days later. Allclones were transferred to 6-well plates and screened by anti-human FcELISA three days later. The six clones with the best productivity weredirectly subcloned from the 6-well plate stage.

Limiting Dilution:

The best parental clones of the second transfection and selection round43-16A10_S1, 43-16A10_S13, 43-16A10_S14, 43-16A10_S19, 43-16A10_S24,43-16A10_S43 were subcloned by limiting dilution. The product quality ofthe twelve best subclones was assessed in SDS-PAGE and Western-Blottingfrom the 24-well stage. No unwanted antibody related side products weredetected.

Three subclones, 43-16-A10-S1-16, 43-16-A10-S24-11, and43-16-A10-S43-14, were selected according to their productivity in6-well plates for the expansion in shake flasks. They were transferredto serum free production medium for the final productivity assessment.Their productivity was compared to the subclone after the firsttransfection, clone 43-16-A10. The productivity was increased twofoldfor two of the clones after the second transfection and selection,43-16-A10-S1-16 and 43-16-A10-S24-11, from 221 μg/ml after 7 days in thebatch shake flask to 436 μg/ml and 407 μg/ml, respectively. After 10days incubation in the batch shake flask the productivity increased from306 μg/ml to 683 μg/ml and 446 μg/ml, respectively.

The specific productivity per cell increased as well from 17 pg/cell/dayfor the clone 43-16-A10 after the first transfection to 40 pg/cell/dayfor the first transfected clone 43-16-A10-S1-16 and to 33 pg/cell/dayfor the second transfected clone 43-16-A10-S24-11. The doubling time wasnot affected by the second transfection. The doubling time for the clone43-16-A10 after the first transfection was 33 h and it was 32 h for bothclones 43-16-A10-S1-16 and 43-16-A10-S24-11.

Example 7

Expression Vector for Expressing an Anti-IL-13Rα Antibody

Another example (preferably monoclonal) antibody for which a cell linefor expression can be obtained according to the current invention is anantibody binding to the IL-13 Receptor alpha 1 (anti-IL-13Rα1anti-IL-13Rα antibody). Such an antibody and the corresponding nucleicacid sequences are for example described in WO 2006/072564 or SEQ ID NO:19 to 28.

A genomic human kappa-light chain constant region gene segment (C-kappa)was added to the light chain variable region of the anti-IL-13Rαantibody whereas a human gamma 1-heavy chain constant region genesegment (C_(H1)-Hinge-C_(H2)-C_(H3)) was added to the heavy chainvariable region of the anti-IL-13Rα antibody. The expression plasmid5321 comprises an expression cassette for the anti-IL-13Rα antibodyγ1-heavy chain, and the anti-IL-13Rα antibody κ-light chain, and anucleic acid encoding the murine DHFR gene. An annotated plasmid map isshown in FIG. 9.

a) Heavy Chain Expression Cassette

The transcription unit of the anti-IL-13Rα antibody conjugate heavychain is composed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (CMV IE),    -   a 5′-untranslated region (5′ UTR),    -   the coding sequence for the anti-IL-13Rα antibody gamma 1-heavy        chain conjugate including a signal peptide in an intron-exon        gene structure,    -   the human gamma 1-immunoglobulin polyadenylation signal        sequence.        b) Light Chain Expression Cassette

The transcription unit of the anti-IL-13Rα antibody light chain iscomposed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (CMV IE),    -   a 5′-untranslated region (5′ UTR),    -   the coding sequence for the anti-IL-13Rα kappa-light chain in an        intron-exon gene structure,    -   the human immunoglobulin kappa-polyadenylation signal sequence.        c) Expression Plasmids

For the expression and production of the anti-IL-13Rα antibody conjugatethe light and heavy chain expression cassettes were placed on a singleexpression vector (light chain upstream of heavy chain). Two identicalexpression vectors were generated differing only in the selectablemarker gene included, in particular, the murine DHFR gene and both themurine DHFR gene and a hygromycin resistance gene.

The expression vectors contain beside the light and heavy chainexpression cassette the following elements:

-   -   an origin of replication allowing for the replication of the        plasmid in E. coli (pUC origin),    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

Example 8

Transfection and Selection of a CHO Cell Line Expressing an Anti-IL-13RαAntibody

For the first transfection and selection step the plasmid 5321 has beenused. Plasmid 5321 has been transfected with electroporation into parentcell line adapted to growth in ProCHO4-complete medium. The transfectedcells were cultivated in HyQSFMCHO-medium (HyClone) supplemented with upto 200 nM methotrexate in plates. The antibody concentration in theculture supernatants was evaluated by an anti-human IgG1 ELISA. Theclones have been tested and the selected of them were further cultivatedin 24-well plates, 6-well plates and subsequently in shaker flasks. Thegrowth and productivity was assessed in static and suspension culturesby anti-human IgG1 ELISA and/or analytic Protein A HPLC. The best clone(best clone does not denote the most productive clone it denotes theclone with the best properties for the further steps) was selected. Theselected clone was named 200_(—)019. Productivity was 90 μg/ml with anaverage specific production rate of 7 pg/cell*d after 7 days ofcultivation.

For the second transfection and selection step a plasmid with a DHFR andhygromycin resistance gene has been used. The plasmid has beentransfected with electroporation into the selected cell line cultivatedin HyQSFMCHO-medium (HyClone) supplemented with up to 200 nMmethotrexate. The double selection medium contained in addition 300μg/ml hygromycine B. Single antibody secreting cells were identified anddeposited on the basis of their fluorescence intensity after stainingwith a Protein A Alexa Fluor conjugate by FACS analysis. The selectedclone was named 5_(—)17_(—)35. Productivity was 150 μg/ml with anaverage specific production rate of 10 pg/cell*d after 7 days ofcultivation.

Example 9

Expression Vector for Expressing an Anti-CD4 Antibody Conjugate

Another example (monoclonal) antibody for which a cell line forexpression can be obtained according to the current invention is anantibody against the human CD4 surface receptor (anti-CD4 antibody)which is conjugated to two to eight antifusogenic peptides. Such anantibody and the corresponding nucleic acid sequences are for examplereported in PCT/EP2008/005894 or SEQ ID NO: 29 to 40.

A genomic human kappa-light chain constant region gene segment (C-kappa)was added to the light chain variable region of the anti-CD4 antibody ofSEQ ID NO: 39, whereas a human gamma 1-heavy chain constant region genesegment (C_(H1)-Hinge-C_(H2)-C_(H3)) was added to the heavy chainvariable region of the anti-CD4 antibody of SEQ ID NO: 36. Theexpression plasmid 6311 comprises an anti-CD4 antibody γ1-heavy chain,which is joint at the last but one C-terminal amino acid, i.e. theC-terminal lysine residue of the heavy chain is removed, with a nucleicacid encoding an antifusogenic peptide of SEQ ID NO: 41 via the peptidicglycine-serine linker of SEQ ID NO: 42, and a anti-CD4 antibody κ-lightchain, and a nucleic acid conferring resistance to the selectable markerneomycin. An annotated plasmid map is shown in FIG. 8.

a) Heavy Chain Expression Cassette

The transcription unit of the anti-CD4 antibody conjugate heavy chain iscomposed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (CMV IE),    -   a 5′-untranslated region (5′ UTR),    -   the coding sequence for the anti-CD4 antibody gamma 1-heavy        chain conjugate including a signal peptide in an intron-exon        gene structure,    -   the SV 40 early poly A signal sequence.        b) Light Chain Expression Cassette

The transcription unit of the anti-CD4 antibody conjugate light chain iscomposed of the following elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (CMV IE),    -   a 5′-untranslated region (5′ UTR),    -   the coding sequence for the anti-CD4 kappa-light chain in an        intron-exon gene structure,    -   the SV 40 early poly A signal sequence.        c) Expression Plasmids

For the expression and production of the anti-CD4 antibody conjugate thelight and heavy chain expression cassettes were placed on a singleexpression vector (light chain upstream of heavy chain). Three identicalexpression vectors were generated differing only in the selectablemarker gene included, in particular, a neomycin resistance gene, apuromycin resistance gene, and a hygromycin resistance gene.

The expression vectors contain beside the light and heavy chainexpression cassette the following elements:

-   -   an origin of replication allowing for the replication of the        plasmid in E. coli taken from pUC18 (pUC origin),    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.

Example 10

Transfection and Selection of a CHO Cell Line Expressing an Anti-CD4Antibody Conjugate

Transfection and Selection Steps:

For the first transfection and selection step the plasmid 6311 has beenused. Plasmid 6311 has been transfected with electroporation into parentcell line adapted to growth in ProCHO4-complete medium. The transfectedcells were cultivated in ProCHO4-complete medium supplemented with up to700 μg/ml G418 in 96 well plates. The antibody concentration in theculture supernatants was evaluated by an anti-human IgG1 ELISA.Approximately 5000 clones have been tested and the selected of them werefurther cultivated in 24-well plates, 6-well plates and subsequently inshaker flasks. The growth and productivity of approximately 15 cloneswas assessed in static and suspension cultures by anti-human IgG1 ELISAand/or analytic Protein A HPLC. The best clone (best clone does notdenote the most productive clone it denotes the clone with the bestproperties for the further steps) was subcloned by limited dilution inProCHO4-conditioned medium supplemented with 700 μg/ml G418.

Subclones were obtained by two methods, Limiting Dilution (LD) andFluorescence Activated Cell Sorting (FACS).

Limiting Dilution:

For limiting dilution cells were plated out in ProCHO4-selection mediumat a cell density of 0.5-2 cells per 0.1 ml medium per well of a 96-wellculture plate.

Single Cell Deposition by Flow Cytometry Including Identification andIsolation of Clones:

In the case of fluorescence activated cell sorting the electroporatedpopulation of cells were directly seeded into T-flasks inProCHO4-complete medium. The appropriate selection agent or agents(G418, hygromycin, and/or puromycin) was/were added to the culture oneday after transfection and the transfectant pool was expanded. Thegrowth and productivity of approximately 112 clones was assessed instatic and suspension cultures by anti-human IgG1 ELISA and/or analyticProtein A HPLC. The selected clone was named I-17.

For the second transfection and selection step a plasmid with ahygromycin resistance gene has been used. The plasmid has beentransfected with electroporation into cell line clone I-17 cultivated inProCHO4-complete medium supplemented with 700 μg/ml G418. Thetransfected cells were expanded for about two to three weeks inProCHO4-conditioned medium supplemented with 200 μg/ml G418 and 300μg/ml hygromycin (ProCHO4-double selection medium). Single antibodysecreting cells were identified and deposited on the basis of theirfluorescence intensity after staining with a Protein A Alexa Fluorconjugate by FACS analysis. The deposited cells were cultivated inProCHO4-double selection medium in 96 well plates. The antibodyconcentration in the culture supernatants was evaluated by an anti-humanIgG1 ELISA. The selected clone was named 24_(—)16. For the thirdtransfection and selection step a plasmid with a puromycin resistancegene has been used. The plasmid has been transfected withelectroporation into cell line clone 24_(—)16 cultivated inProCHO4-double selection medium. The transfected cells were expanded forabout two to three weeks in ProCHO4-triple selection medium(ProCHO4-conditioned medium supplemented with 200 μg/ml G418 and 300μg/ml hygromycin and 4 μg/ml puromycin). Single antibody secreting cellswere identified and deposited on the basis of their fluorescenceintensity after staining with a Protein A Alexa Fluor conjugate by FACSanalysis. The deposited cells were cultivated in ProCHO4-tripleselection medium in 96 well plates. The antibody concentration in theculture supernatants was evaluated by an anti-human IgG1 ELISA. Theselected clone was named 1_(—)24.

Clone Characteristics:

As can be seen from the following table the doubling time and the celldensity after three days of cultivation were comparable when the basiccell line CHO-K1 (wild-type) and the selected clones are compared.

TABLE 5 Growth characteristics Doubling Starting Cell density Viabilitytime cell density at day 3 at day 3 Clone [h] [10⁶ cells/ml] [10⁶cells/ml] [%] CHO-K1 22-25 3 18-22 96-98 (pre adapted) I-17 25-30 313-15 95-97 24_16 25-30 3 15-16 95-96 1_24 30-32 3 12-14 95-97

The invention claimed is:
 1. A method for the recombinant production ofa heterologous immunoglobulin in a CHO cell which is secreted to thecultivation medium comprising: a) providing a CHO cell, wherein said CHOcell is adapted to growth in suspension culture, adapted to growth inserum-free medium, and mycoplasma free, b) providing a nucleic acidcomprising i) a prokaryotic origin of replication, ii) a first nucleicacid sequence conferring resistance to a prokaryotic selection agent,iii) a second nucleic acid sequence encoding the heavy chain of saidheterologous immunoglobulin, and a third nucleic acid sequence encodingthe light chain of said heterologous immunoglobulin, whereby a firsttransfection vector is provided which comprises said provided nucleicacid and an additional fourth nucleic acid sequence conferringresistance to a first eukaryotic selection agent, whereby a secondtransfection vector is provided which comprises said provided nucleicacid and an additional fourth nucleic acid sequence conferringresistance to a second eukaryotic selection agent, whereby said secondeukaryotic selection agent is different to said first eukaryoticselection agent, b1) providing a nucleic acid comprising i) aprokaryotic origin of replication, ii) a first nucleic acid sequenceconferring resistance to a prokaryotic selection agent, iii) a secondnucleic acid sequence encoding the heavy chain of said heterologousimmunoglobulin, and/or a third nucleic acid sequence encoding the lightchain of said heterologous immunoglobulin, whereby a third transfectionvector is provided which comprises said provided nucleic acid and anadditional fourth nucleic acid sequence conferring resistance to a thirdeukaryotic selection agent, whereby said third eukaryotic selectionagent is different to said first eukaryotic selection agent and is alsodifferent to said second eukaryotic selection agent, c) transfectingsaid CHO cell, wherein said transfecting comprises the following: (i)transfecting said CHO cell with said first transfection vector, (ii)selecting a CHO cell transfected in (i) by selected growth incultivation medium containing a first eukaryotic selection agent towhich the first transfection vector confers resistance, (iii)transfecting said selected CHO cell in (ii) with said secondtransfection vector, (iv) selecting a CHO cell transfected in (iii) byselected growth in cultivation medium containing said first eukaryoticselection agent to which the first transfection vector confersresistance and said second eukaryotic selection agent to which thesecond transfection vector confers resistance, (v) transfecting said CHOcell selected in (iv) with said third transfection vector, (vi)selecting a CHO cell transfected in (v) by selected growth in acultivation medium containing said first eukaryotic selection agent towhich the first transfection vector confers resistance and said secondeukaryotic selection agent to which the second transfection vectorconfers resistance and said third eukaryotic selection agent to whichthe third transfection vector confers resistance, d) cultivating saidtransfected CHO cell in a medium in the presence of said first and saidsecond and third eukaryotic selection agent, under conditions suitablefor the expression of said second, and/or third nucleic acid, and e)recovering said secreted heterologous immunoglobulin from thecultivation medium and thereby producing a heterologous immunoglobulinin a CHO cell which is secreted to the cultivation medium; wherein saidresultant CHO cell is stable in the absence of any or all selectionagents, as used in the previous steps, for up to generation
 60. 2. Themethod of claim 1, wherein said CHO cell is selected from the groupconsisting of a CHO K1 cell, a CHO DG44 cell, a CHO XL99 cell, a CHODXB11 cell, and a CHO DP12 cell; and wherein further the heterologousimmunoglobulin is selected from the group consisting of an anti-ABantibody, an anti P-selection antibody, an anti-IL-13R or antibody andan anti-CD4 antibody conjugate.
 3. The method of claim 2, wherein saidsecond and/or third nucleic acid contains hybrid intronic nucleic acidsequence.
 4. The method of claim 2, characterized in that said firsttransfection vector and said second transfection vector differ only inthe nucleic acid conferring resistance to said eukaryotic selectionagent.
 5. The method of claim 2, wherein step c) and step d) areperformed in the same medium.
 6. The method of claim 5, wherein saidmedium is selected form the group consisting of a serum-free medium, aserum-free medium supplemented with defined animal-derived components,an animal-derived component free medium, a protein-free medium, aprotein-free medium supplemented with defined animal-derived components,a defined protein-free medium, and a chemically defined medium.
 7. Themethod of, claim 2, wherein the cultivating of step d) is either in thepresence of the eukaryotic selection agents in a volume of less than 500liter or said cultivating is in the absence of said eukaryotic selectionagents in a volume of 500 liter or more, and that the recovering of thesecreted heterologous immunoglobulin is from the cultivation mediumwithout said eukaryotic selection agents.
 8. The method of claim 2,characterized in that the productivity of said CHO cells is over 40generations not less than 70% and not more than 130% of the productivityafter 10 generations of cultivation as split-batch cultivation.
 9. Themethod of claim 8, characterized in that the productivity of said CHOcell is at least 1.5 g/I of said heterologous immunoglobulin within 21days as fed-batch cultivation.
 10. The method of claim 1, characterizedin that said method further comprises: f) purifying said heterologousimmunoglobulin with one or more chromatographic steps.
 11. The method ofclaim 10, characterized in that said transfected CHO cell of step c) hasa doubling time of 150% or less of the doubling time of the CHO cellselected in substep (ii), a volumetric yield of at least 125% comparedto the volumetric yield of the CHO cell selected in (ii).